WO2017036294A1 - 马克斯克鲁维酵母菌及其应用 - Google Patents

马克斯克鲁维酵母菌及其应用 Download PDF

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WO2017036294A1
WO2017036294A1 PCT/CN2016/094868 CN2016094868W WO2017036294A1 WO 2017036294 A1 WO2017036294 A1 WO 2017036294A1 CN 2016094868 W CN2016094868 W CN 2016094868W WO 2017036294 A1 WO2017036294 A1 WO 2017036294A1
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marxianus
gene
strain
inulinase
animal feed
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French (fr)
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吕红
余垚
周峻岗
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Fudan University
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Fudan University
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Priority claimed from CN201510540030.6A external-priority patent/CN105087403B/zh
Priority claimed from CN201510540029.3A external-priority patent/CN105112313B/zh
Priority claimed from CN201510562496.6A external-priority patent/CN105063080A/zh
Priority claimed from CN201510562498.5A external-priority patent/CN105132452A/zh
Priority claimed from CN201510562564.9A external-priority patent/CN105063081A/zh
Priority claimed from CN201510562932.XA external-priority patent/CN105063082A/zh
Priority claimed from CN201610058751.8A external-priority patent/CN105707436A/zh
Application filed by Fudan University filed Critical Fudan University
Publication of WO2017036294A1 publication Critical patent/WO2017036294A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to a yeast, in particular to a K. marxianus, K. marxianus expression system having high secretion performance and high biomass, and application thereof, and to the expression of the yeast Enzymes/proteins, and the use of the above substances in the fields of feed, feed additives, foods, vaccines, drugs, and the like.
  • Kluyveromyces marxianus (K. marxianus) is ubiquitous in nature in Khrisanfova L P, Antimicrobial properties of Koumiss from cow and mare milk, Moloch. Prom., 1969, 30(10): 16 ), yogurt (Fleet GH, Spoilage yeasts, Crit Rev Biotechnol., 1992, 12: 1-44; Mandarin A et al, Food spoilage yeasts, In Rose AH, Harrison JS (eds) The yeasts, vol 5, 2nd edn. Academic Press, London, 1993: 435-515.), Milk (Wendel B.
  • the present application provides a K. marxianus, Mark with high secretion performance and high biomass.
  • S. cerevisiae expression system enzyme/protein expressed by the yeast, and application in the fields of feed, feed additives, food, vaccines, drugs, and the like.
  • a first aspect of the present invention provides a Kluyveromyces marxianus (K. marxianus) having the gene sequence of SEQ ID No. 1, or SEQ ID No. 1 A gene sequence with a degree of homology of at least 90%.
  • K. marxianus having the gene sequence of SEQ ID No. 1, or SEQ ID No. 1 A gene sequence with a degree of homology of at least 90%.
  • the K. marxianus has a degree of homology to SEQ ID No. 1 of at least 93%, preferably at least 95%, more preferably at least 98%, more preferably at least 99%.
  • the genetic sequence is a degree of homology to SEQ ID No. 1 of at least 93%, preferably at least 95%, more preferably at least 98%, more preferably at least 99%.
  • the K. marxianus preservation number is CGMCC No. 10621 (in the following content of the invention, it may also be referred to as K. marxianus FIM-1, K. marxianus FIM- 1 or Kluyveromyces marxianus FIM-1).
  • the K. falciparum CGMCC No. 10621 of the present invention is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee; the address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing The deposit date is March 13, 2015.
  • the present invention has the colony of K. falciparum CGMCC No.10621, which has a round, milky white color and a neat edge; the yeast is elliptical, and the size is about 4-8 ⁇ m, budding reproduction, single, two or more. String adhesion.
  • the K. marxianus yeast of the invention ferments and secretes endo-inulinase, and the yield of inulinase is greater than that of glucose and inulin mixed carbon source under the condition of using glucose as a carbon source.
  • a second aspect of the present invention provides a yeast expression system constructed by K. marxianus, preferably a recombinant expression system of a foreign protein.
  • the yeast expression system preferably a recombinant expression system of a foreign protein, may be any one or more of the gene mutant strain of K. marxianus, a strain obtained by genetic engineering means, and the gene mutation
  • the strain obtained by the strain, genetic engineering means has a biomass similar to that of the K. marxianus described in the first aspect of the invention.
  • the "similar” means that the difference in biomass data between the K. marxianus strains according to the first aspect of the present invention is ⁇ 5%, more preferably ⁇ 3%.
  • K. marxianus is preferably the Markskruvi described in the first aspect of the invention
  • the yeast is more preferably K. marxianus CGMCC No. 10621.
  • the present invention provides an auxotrophic strain of K. marxianus, that is, a strain after knocking out all or part of a nutritional gene.
  • the nutritional gene is preferably any one or more selected from the group consisting of a URA3 gene, a HIS3 gene, and an ADE2 gene.
  • the K. marxianus strain knocks out all or part of the URA3 gene.
  • the knockout partial gene URA3 gene preferably knocks out at least all or part of the ORF region of the gene, and more preferably at least knocks out all or part of the CDS region of the gene.
  • the K. marxianus auxotrophic strain cannot grow in Uracil auxotrophic medium.
  • the skeletal bacteria of the K. marxianus auxotrophic strain according to the present invention may further be a strain after the K. marxianus strain knocks out one or more of all or part of the HIS3 gene and the ADE2 gene.
  • the K. marxianus auxotrophic strain cannot grow in the SC-Histidine auxotrophic medium after knocking out all or part of the HIS3 gene.
  • the K. marxianus auxotrophic strain cannot grow in SC-Adenine auxotrophic medium after knocking out all or part of the ADE2 gene.
  • the K. marxianus auxotrophic strain is a strain in which the K. marxianus strain knocks out all or part of the URA3 gene, and all or part of the HIS3 gene.
  • the K. marxianus auxotrophic strain is unable to grow in SC-Uracil, SC-Histidine auxotrophic medium.
  • the K. marxianus auxotrophic strain is a strain in which the K. marxianus strain knocks out all or part of the URA3 gene, and all or part of the ADE2 gene.
  • the K. marxianus auxotrophic strain is unable to grow in SC-Uracil, SC-Adenine auxotrophic medium.
  • the K. marxianus auxotrophic strain is a strain in which the K. marxianus strain knocks out all or part of the URA3 gene, all or part of the HIS3 gene, and all or part of the ADE2 gene.
  • the K. marxianus auxotrophic strain is unable to grow in SC-Uracil, SC-Histidine, SC-Adenine auxotrophic medium.
  • the K. marxianus auxotrophic strain is a strain after knocking out all or part of the HIS3 gene by the K. marxianus strain.
  • the knock-out portion HIS3 gene is an ORF region that at least knocks out all or part of the gene, more preferably a CDS region that knocks out at least all or part of the gene.
  • the K. marxianus auxotrophic strain is unable to grow in SC-Histidine auxotrophic medium.
  • the K. marxianus auxotrophic strain is a strain in which the K. marxianus strain knocks out all or part of HIS3, and all or part of the ADE2 gene.
  • the Marker's yeast auxotrophic strain is unable to grow in SC-Histidine, SC-Adenine auxotrophic medium.
  • the present invention also provides a yeast expression system constructed by Kluyveromyces cerevisiae, more preferably a strain which knocks out the inulinase gene of K. marxianus; or the auxotrophic strain of K. marxianus described above. A strain that knocks out the inulinase gene.
  • K. marxianus is preferably the K. marxianus strain according to the first aspect of the invention, more preferably Kluyveromyces cerevisiae CGMCC No. 10621.
  • a third aspect of the present invention provides a recombinant vector for secretory expression of a foreign gene in an auxotrophic strain of K. marxianus.
  • the K. marxianus is preferably the K. marxianus strain according to the first aspect of the invention, more preferably Kluyveromyces cerevisiae CGMCC No. 10621.
  • the recombinant vector comprises, in order, a PKD1 vector, an inulinase promoter, a signal peptide, a multiple cloning site, an inulinase terminator, a nutrient gene promoter, and a nutrition.
  • Gene open reading frame (ORF) ORF
  • the recombinant vector comprises, in order, a PKD1 vector, an inulinase promoter, a multiple cloning site, an inulinase terminator, a nutrient gene promoter, and a nutrient gene open reading. Box (ORF).
  • the recombinant vector comprises, in order, a PKD1 vector sequence, an inulinase promoter, a multiple cloning site, an inulinase terminator, a nutrient gene promoter, and a nutrition.
  • An open reading frame (ORF) of the gene wherein the recombinant vector further comprises a polyhistidine tag located at the C-terminus or the N-terminus of the multiple cloning site.
  • the recombinant vector of the third aspect of the invention may or may not include an ampicillin resistance gene; preferably, if an ampicillin resistance gene is present, the PKD1 is located in the order of the ampicillin resistance gene and the inulinase activation Between the children. More preferably, the recombinant vector does not comprise an ampicillin resistance gene.
  • the signal peptide is preferably any one or more of an inulinase signal peptide or a yeast alpha factor signal peptide, and is preferably an inulinase signal peptide.
  • the recombinant vector comprises, in order, a PKD1 vector, an inulinase promoter, an inulinase signal peptide, a multiple cloning site, an inulinase terminator, a nutrient gene promoter, and a nutrient gene opening. Reading frame (ORF).
  • polyhistidine tag is preferably a tetrameric or higher histidine tag, more preferably a pentameric or higher histidine tag, more preferably a hexameric or higher histidine tag.
  • the nutritional gene is preferably a nutritional gene of a Marks Budapest strain, such as any one or more of a URA3 gene, a HIS3 gene, and an ADE2 gene, and more preferably a URA3 gene.
  • the multiple cloning site may be a sequence comprising one or more restriction enzyme sites, such as any one or several of Smal, Xmal, Spel, Notl More preferably, it is any one or more of Spel, Notl, and Smal and/or Xmal, such as Smal/Xmal, Spel, Notl.
  • the multiple cloning site length is preferably 15-25 bp, more preferably 18-23 bp, and still more preferably 19-21 bp.
  • the DNA sequence of the recombinant vector is selected from any one or more of SEQ ID No. 41-SEQ ID No. 46, SEQ ID No. 89.
  • the present invention also provides a transformant which transfers any one or more of the recombinant vectors of the third aspect of the present invention into a strain of K. marxianus, preferably into any of the above-mentioned gene-deficient types of the present invention.
  • a strain of K. marxianus it is more preferably a strain which is transferred into the URA3 gene-deficient K. marxianus.
  • the K. marxianus is preferably the K. marxianus strain according to the first aspect of the invention, more preferably Kluyveromyces cerevisiae CGMCC No. 10621.
  • a fourth aspect of the present invention provides a use of K. marxianus in product fermentation or product additive, wherein the product may be any one of animal feed, animal feed additive, food, vaccine, and medicine. Or several, and preferably used as a fermenting agent, such as animal feed additives and / or moving Feed feed fermenting agent.
  • the use of the K. marxianus in the feed fermentation or feed additive of the present invention can improve animal immunity, and/or improve animal digestion, and/or protect animals from free radical damage, and/or produce more a nutrient component; more preferably, it has superior activity in improving animal immunity, and/or improving animal digestion, and/or protecting animals from free radical damage, and/or producing more nutrients than existing yeasts. ability.
  • K. marxianus is preferably a yeast expression system constructed by the K. marxianus according to the first aspect of the invention, and/or the K. marxianus described in the second aspect, And/or the transformant of the third aspect of the invention.
  • the K. marxianus extract of the present invention is used as an animal feed additive and/or an animal feed fermenting agent, which can improve the immunity of the animal and/or improve the feed compared with the existing K. marxianus yeast.
  • Animals have the ability to digest, and/or protect animals from free radical damage, and/or the ability to produce more nutrients.
  • the fermenting agent is directly added to the animal feed additive and/or the animal feed, the food, the vaccine or the medicine, or B) the fermenting agent can be fermented to prepare the raw material.
  • the Marker's yeast of the present invention is used as an animal feed additive and/or an animal feed fermenting agent, which can improve animal immunity, such as: promoting T cell function, promoting macrophage function, And provide stronger sugar chain immune promotion.
  • the K. marxianus extract of the present invention is used as an animal feed additive and/or an animal feed fermenting agent, which enables the animal to avoid or reduce D-lactic acid poisoning, such as: capable of reducing or eliminating D- Lactic acid.
  • the K. marxianus extract of the present invention is used as an animal feed additive and/or an animal feed fermenting agent capable of reducing or eliminating lactose intolerance.
  • the K. marxianus extract of the present invention is used as an animal feed additive and/or an animal feed fermenting agent capable of degrading lipids and reducing or eliminating the accumulation of lipids in the animal digestive system.
  • the Marker's yeast of the present invention is used as an animal feed additive and / or animal feed fermentation bacteria, can provide the ability to eliminate free radicals, protect animals from free or less damage by free radicals. For example, it is possible to reduce peroxide in animals.
  • the K. marxianus extract of the present invention is used as an animal feed additive and/or an animal feed fermenting agent capable of providing biotin and/or protein to an animal.
  • the invention also provides an additive, which may be any one or several of animal feed, food, vaccine, and pharmaceutical additive terminals.
  • the additive comprises at least A) the K. marxianus strain of the first aspect of the invention and the enzyme/protein thereof, and/or the yeast constructed by the K. marxianus described in the second aspect.
  • the invention also provides a product, which may be any one or several of animal feed, vaccine, medicine, food, including at least one additive, the at least one additive comprising A) the first invention
  • the yeast expression system constructed by the K. marxianus and its expression enzyme/protein, and/or B) of the second aspect of the invention, and the expression enzyme/protein, and/or Or C) any one or more of the transformant of the third aspect of the invention and the expression enzyme/protein thereof.
  • the present invention also provides a fermenting agent comprising at least the Marker's yeast according to the first aspect of the invention, and/or the Markskruvi described in the second aspect A yeast expression system constructed by yeast, and/or a transformant of the third aspect of the invention.
  • the present invention also provides a fermented product, which may be selected from any one or more of animal feed, food, vaccine or medicine, and the fermented product is obtained by fermenting the raw material in the presence of a fermenting agent, wherein
  • the fermenting agent comprises at least the yeast expression system constructed by the K. marxianus described in the first aspect of the invention, and/or the K. marxianus described in the second aspect, and/or the invention The transformant described in the third aspect.
  • any of the above products, and/or product additives of the present invention may further comprise nutrients, flavoring agents, other beneficial bacteria, antifungal agents, bile acids, chelating peptides, complex enzymes, growth promoting components, antibiotics, Any one or more of pigments, and other components that can be used in products or product additives.
  • the fermenting agent may further include other yeasts usable for fermentation.
  • the vaccine or the drug of the present invention may be a vaccine or a drug for use in humans or animals.
  • the animal of the present invention may be any one or more of the following animals: A) spines Invertebrates such as phylum, arthropods, and mollusks; B) vertebrates such as fish, amphibians, reptilian, avian, and mammalian.
  • A) spines Invertebrates such as phylum, arthropods, and mollusks
  • B) vertebrates such as fish, amphibians, reptilian, avian, and mammalian.
  • snails such as snails, snails, abalone, shellfish, shrimp, crab, sea cucumber, sea urchin, fish, frog, snake, turtle, pig, cow, sheep, horse, dog, cat, mouse, rabbit, donkey, donkey, dolphin, shark , meat animals such as whales, chickens, ducks, geese, pelicans, pigeons, ostriches, egg-type animals, medicinal animals, fur animals, ornamental animals, and other economic animals.
  • the Marker's yeast of the present invention may be preferably the K. marxianus described in the first aspect of the present invention, unless otherwise specified, and more preferably Kluyverver's yeast CGMCC No.10621.
  • the K. marxianus provided by the invention has higher ability to secrete protein and higher biomass than the existing Kluyveromyces yeast such as ATCC8585 and ATCC26548, and can be used for producing an enzyme preparation, and can be used To construct a recombinant protein expression system.
  • the auxotrophic K. marxianus strain of the present invention can be used for a host strain of the K. marxianus expression system for recombinant preparation of a foreign protein (enzyme).
  • K. marxianus as a feed additive can improve animal immunity more than Saccharomyces cerevisiae, can provide more protein and biotin, and can detoxify D-lactic acid at the same time; It was found that the K. marxianus, especially the K. falciparum CGMCC No. 10621 strain, can provide more protein and help the animal to resist oxidation compared with the other five reported K. marxianus. It also metabolizes lactose and lipids and has advantages in expression systems that secrete foreign proteins.
  • Figure 1 is the ability of K. marqueus to secrete proteins of the present invention
  • Lane 1 K. marxianus FIM-1 of the present invention
  • Lane 2 K. marxianus ATCC 26548
  • Lane 3 K. lactis ATCC8585
  • Lane M Protein Marker ;
  • Figure 2 is a cell morphology of K. marxianus according to the present invention.
  • Figure 3 is a high biomass fermentation result of the K. marqueus yeast of the present invention.
  • Figure 4 is a schematic diagram showing the deletion of K. falciparum CGMCC No.10621 gene in other K. marxianus strains, and the related gene of CGMCC No.10621 (FIM-1) is shown in the left frame. The gray in the square indicates that the corresponding strain of the gene does not exist; wherein, Figure 4A is the protein. Genes are generated, Figure 4B shows genes related to rapid cell growth, and Figure 4C shows other genes such as antioxidant capacity, lactose metabolism, lipid metabolism, and protein secretion process genes;
  • CCT means K.maxcianus CCT7735
  • NBRC means K.maxcianus NBRC1777
  • KCTC means K.maxcianus KCTC17555
  • DMB1 means K.maxcianus DMB1K
  • DMKU means K.maxcianus DMKU3-1042;
  • Figure 5 is a schematic diagram showing the construction of pMD-18T ura3 ⁇ -cas plasmid
  • Figure 6 is a schematic diagram showing the knockdown of the URA3 gene on the FIM genome by homologous recombination method
  • Figure 7 is a graph showing the growth state of the obtained FIM-1 (ura3 ⁇ ) and wild-type FIM on a uracil auxotrophic medium;
  • Figure 8 is a schematic diagram showing the construction of pMD-18T his3 ⁇ -ca plasmid
  • Figure 9 is a schematic diagram showing the construction principle of FIM-2 (ura3 ⁇ his3 ⁇ ) strain
  • Figure 10 is a result of the screening process of the FIM-2 (ura3 ⁇ his3 ⁇ ) strain construction
  • Figure 11 is a schematic diagram showing the construction of pMD-18T ade2 ⁇ URA3 plasmid
  • Figure 12 is a panel identification result of FIM-4 (ura3 ⁇ ade2 ⁇ ) strain
  • Figure 13 is a panel identification result of FIM-5 (ura3 ⁇ his3 ⁇ ade2 ⁇ ) strain
  • Figure 14 shows the results of plate identification of FIM-3 (his3 ⁇ ) strain.
  • 15 is a schematic diagram of a reconstructed carrier PUKD112 constructed in an embodiment of the present invention.
  • FIG. 16 is a schematic diagram of a recombinant carrier PUKD117 constructed in Embodiment 2 of the present invention.
  • FIG. 17 is a schematic diagram of a recombinant carrier PUKD115 constructed in an embodiment of the present invention.
  • FIG. 18 is a schematic diagram of a reconstructed carrier PUKD118 constructed in an embodiment of the present invention.
  • 19 is a schematic diagram of a reconstructed carrier PUKD 114 constructed in an embodiment of the present invention.
  • FIG. 20 is a schematic diagram of a reconstructed carrier PUKD116 constructed in an embodiment of the present invention.
  • yeast solid medium YNB no amino A yeast nitrogen source; Yeast Nitrogen Base without Amino Acids
  • SDS-PAGE protein electrophoresis-SDS polyacrylamide gel electrophoresis
  • yeast FIM-1 has a high secreted protein capacity (as shown in Figure 1), and the secreted protein has a size of about 90 kDa.
  • the strain of yeast FIM-1 was identified using the 18s rDNA method.
  • yeast FIM-1 Using the genome of yeast FIM-1 as a template, 18S rDNA universal primers NS1 (gtagtcatatgcttgtctc) and NS8 (tccgcaggttcacctacgga) were used for PCR amplification and sequencing.
  • the 18s rDNA sequence was obtained as shown in SEQ ID NO.1, and GenBank. After the known sequences in the alignment, the 18s rDNA sequence of the obtained yeast FIM-1 was found to have a sequence similarity to K. marxianus NBRC1777 of 100%.
  • the Kluyveromyces cerevisiae according to the present invention is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with a preservation date of On March 13, 2015, the deposit number was CGMCC No.10621.
  • Fermentation process control pH 7.0, adjusted with 30% ammonia water, and adjusted with 10% HCl; dissolved oxygen is set to 30% DOT, and dissolved oxygen is adjusted by the rotation speed.
  • Fermentation culture time 48-72 hours.
  • Culture temperature 28-35 ° C.
  • the feed flow rate is 20-35g/h glucose flow rate for carbon source and 1-1.25g/h nitrogen source feed
  • the fermentation cycle is 48- At 96 hours, the total fermentation broth was about 2.2-3.5 L when placed.
  • the total amount of glucose consumed in the fermentation process is 850-1110 g
  • the consumption of yeast extract is about 180-232 g
  • the biomass of yeast cells is about 78-117 g/L (dry weight)
  • the cell yield is 0.56-0.62
  • the maximum growth rate of cells It is 2.92-3.29g/L.
  • the cell density and cell biomass obtained by feeding at 20g/L glucose flow rate were the highest. As shown in Fig. 3, the cell biomass was 110.1g/L at 36h, and the cell biomass reached 117.2g/L at 57h. Heavy), the cell yield during the fermentation was 0.62.
  • Figure 4 is constructed based on the long sequence of the gene composition of 15 strains of yeast.
  • the 15 strains of yeast are as follows:
  • the C part of the table is the Yarrowia lipolytica yeast in the family Bisporum, and the Pichia pastoris yeast in the Favreaceae family, which differentiated earlier in the yeast and then evolved independently.
  • Sections B and A of the table are all yeasts, commonly referred to as "Saccharomyces complex".
  • the yeast in the form B is closely related to the evolution of the genus Saccharomyces, and often appears in the winemaking process;
  • the yeast in the table A is more closely related to the genus Kluyveromyces, and the genus Kluyveromyces is mostly located in dairy products.
  • the Eremothecium genus is mostly located in cotton cultivation.
  • a-1,3-mannan can promote T cell function
  • S. cerevisiae yeast cell wall glycoprotein N-oligosaccharide chain a-1,3 linked mannose group has higher immunogenicity, can increase the release of cytokines, increase the concentration of interleukin IL-2 to promote T Cell proliferation and differentiation, and enhance interferon activity, thereby improving the animal's non-specific immunity to diseases such as bacteria.
  • Table 1 and Table 2 show the number of key repeat genes in yeast and between species, Table 1 shows the number of repeat genes in 15 yeast groups, and Table 2 shows 6 strains in K. marxianus (KM). The number of yeast repeat genes.
  • the monosaccharides of ⁇ -glucan in the yeast cell wall are connected by ⁇ -1,3 bond or ⁇ -1,6 bond.
  • This special configuration has a strong stimulating effect on the animal immune system and can directly produce a large number of macrophages. Cells that remove body damage, aging self-cells, and pathogenic microorganisms that invade the body.
  • Figure 4 shows the deletion of the K. marxianus CGMCC No.10621 gene in other K. marxianus strains.
  • the gray square in the figure represents a gene in K. marxianus CGMCC No.10621 in the corresponding other K. marxianus strain. Missing.
  • Part of Figure 4A is mainly related to protein production
  • Part 4B is mainly related to the energy production of mitochondria and the rapid growth of K. marxianus cells.
  • the part of Figure 4C is related to antioxidant capacity, lactose metabolism, lipid metabolism, and protein secretion process.
  • a subunit STT3 of the N-glycosylation initiation step oligosaccharide core transferase in K. marxianus CGMCC No. 10621 is deleted in K. marxianus NBRC1777 (bottom of Figure 4C), which means K.marxianus It has stronger sugar chain immune promoting function than K.marxianus NBRC1777.
  • Lactic acid is divided into L-lactic acid and D-lactic acid. Humans and animals can consume L-lactic acid, but cannot metabolize D-lactic acid. Excessive intake can cause adverse reactions such as metabolic disorders. In yoghurt and lactic acid beverages, mainly L-lactic acid, only a small amount of D-lactic acid.
  • D-lactate dehydrogenase which converts D-lactic acid to pyruvate
  • D-lactate dehydrogenase which converts D-lactic acid to pyruvate
  • the gene DLD encoding D-lactate dehydrogenase generally has a much higher copy number in the Kluyveromyces genus than other yeast species, which means K.marxianus There is greater capacity in D-lactate consumption to protect animals from D-lactic acidosis.
  • lactose intolerance that is, when lactase is absent, the ingested lactose is retained in the intestines and is fermented by harmful bacteria in the intestine to produce acetic acid, propionic acid, butyric acid, etc.
  • Gases include CH 4 , H 2 , CO 2 , causing bloating and diarrhea.
  • the gene ACBP involved in the transport of fatty acyl-CoA in lipid degradation was deleted in K. marxianus NBRC1777 (shown in the lower right panel of Figure 4C), which means that the ability of K. marxianus CGMCC No.10621 to degrade lipids may be Stronger to prevent excessive lipids in the food from accumulating in the intestinal tract of the animal and causing diarrhea.
  • K. marxianus CGMCC No. 10621 Some genes involved in the redox reaction equilibrium to eliminate peroxide in K. marxianus CGMCC No. 10621, and each of the other five K. marxianus yeasts were deleted (middle part of the right side of Fig. 4C). Since the coenzyme NAD+ and NADH are involved in the redox balance, NADP and NADPH can replenish the glutathione in the cell, and other genes directly eliminate the peroxide, so K.marxianus CGMCC No.10621 and other K.marxianus strains In comparison, it has a stronger ability to eliminate free radicals, that is, it has a stronger ability to protect animals from free radical damage.
  • Biotin in B vitamins plays an important role in promoting carboxylation in cells, such as carboxylation of pyruvate in mitochondria to form oxaloacetate, thereby feeding the TCA cycle. Animals cannot synthesize biotin by themselves. In addition to food sources (such as milk, strawberries, grapes, etc.), there are also some colonies from the intestines.
  • BIOA encoding adenosylmethionine-8-amino-7-oxophthalate transaminase
  • BIOD encoding desthiobiotin synthase
  • K. marxianus CGMCC No.10621 Within the K. marxianus species, as shown in Figure 4A, some genes involved in pre-mRNA processing and some ribosomal proteins in K. marxianus CGMCC No.10621 are deleted in strain NBRC1777 and strain DMKU3-1042.
  • K.marxianus CGMCC No.10621 The gene YOR378W on the cell membrane responsible for the high concentration of ammonia inside the cell was deleted in strain CCT7735 and strain NBRC1777. Due to the maturation of mRNA and the efficiency of ribosomes, protein production is affected. The high concentration of ammonia in the cells also indirectly indicates that the organic nitrogen source in the cells is sufficient, and sufficient raw materials are prepared for protein synthesis. Therefore, K. Marxianus CGMCC No.10621 has certain advantages in terms of the amount and efficiency of protein synthesis compared to the existing K. marxianus strains CCT7735, NBRC1777, and DMKU3-1042.
  • K. marxianus CGMCC No.10621 some of the mitochondrial function and energy-generating genes involved in K. marxianus CGMCC No.10621 are mainly deleted in strain NBRC1777, and also in the strain KCTC17555; and K.marxianus CGMCC No.10621 Some genes involved in chromosome segregation and cytoskeleton are also deleted in strain K. marxianus NBRC1777. This means that K.marxianus CGMCC No.10621 has a faster growth rate and therefore provides faster cellular protein.
  • K. marxianus CGMCC No.10621 the genes involved in the protein secretion pathway in K. marxianus CGMCC No.10621 (including the involvement of the nascent peptide chain into the ER, the cleavage signal peptide, the ER to the Golgi transport, and the final exocytosis process), Deletions exist in both K. marxianus strains NBRC1777 and DMKU3-1042, which means that K. marxianus CGMCC No.10621 is more perfect in the protein secretion pathway, ie also as a foreign protein expression system.
  • yeast KGMCC No.10621 has the function of promoting animal immunity, providing abundant protein and biotin for animal growth, and helping to remove harmful D-lactic acid, lactose, and free radicals, and can be used for Animal feed addition and / or feed fermentation.
  • the URA3 gene sequence information of K. marxianus was obtained by NCBI database search, and the primers URA3F (SQE ID No. 2) and URA3R (SQE ID No. 3) were designed to obtain the FIM URA3 gene sequence of 3123 bp.
  • primers URA upstream F (SQE ID No. 4), URA3 upstream R (SQE ID No. 5), and URA3 downstream F (SQE ID No) are designed upstream and downstream of URA3 CDS (Chromosome 1: 543214-1544017, 804 bp), respectively. .6), URA3 downstream R (SQE ID No. 7).
  • a 930 bp DNA fragment upstream of URA3 CDS (Chromosome 1: 154 868-1542797, 930 bp) and a downstream 944 bp DNA fragment (Chromosome 1: 154 046 - 1 544 992, 944 bp) were obtained by PCR amplification.
  • the pMD-18T empty plasmid was digested with Pstl and Smal, and the obtained linearized vector fragment was recovered.
  • the above three fragments were ligated by one-step cloning to obtain a disruption cassette pMD-18T ura3 ⁇ -cas of the URA3 gene knockout.
  • the ura3 ⁇ cassette linear DNA fragment was homologous recombined with the K. marxianus CGMCC No.10621 genomic sequence to achieve knockout of ura3 on the genome.
  • the URA3 deletion mutant plasmid pMD-18T ura3 ⁇ -cas was used as a template, and the deletion mutant fragment ura3 ⁇ cassette linear DNA was obtained by PCR amplification.
  • the fragment was transformed into K. marxianus FIM wild strain, and cultured on a YEPD plate containing 0.2% 5-FOA for 30 hours at 30 ° C, the positive bacteria resistant to 5-FOA were screened.
  • HIS3 up-F SEQ ID No. 8 HIS3 up-R SEQ ID No. 9 HIS3 dow2-F SEQ ID No. 10 HIS3 dow2-R SEQ ID No.11 KmURA3-F SEQ ID No. 12 KmURA3-R SEQ ID No.13 HIS3 dow1-F SEQ ID No.14 HIS3 dow1-R SEQ ID No. 15 HIS3 dow1-KmURA3-F SEQ ID No.16 HIS3 dow1-KmURA3-R SEQ ID No.17
  • the pMD-18T empty plasmid was simultaneously digested with Pstl and Smal, and the obtained linearized vector was recovered.
  • Each of the above DNA fragments was ligated to the vector by a one-step cloning method three times to obtain a pMD-18T his3 ⁇ -cas plasmid.
  • the pMD-18T plasmid ligated with Km URA3 and HIS3 dow1 fragments was amplified by PCR to obtain the HIS3dow1-Km URA3 DNA fragment, wherein the 5' end of the HIS3 dow1-Km URA3 DNA fragment and the 3' end of HIS3up were 10-25 bp homologous sequence, 10' bp homologous sequence at the 3' end of HIS3 dow1-Km URA3 DNA fragment and 5' end of HIS3 dow2, HIS3 dow1-Km DNA fragment and linearization of HIS3 up+HIS3 dow2 DNA fragment
  • the vector was ligated by one-step cloning, and the ligated product was transformed into competent E.
  • the pMD-18T his3 ⁇ -cas plasmid of cassette (HIS3 up-HIS3 dow1-Km URA3-HIS3 dow2, 4-segment DNA fragment).
  • the first fragment was HIS3 up (SEQ IN No. 18)
  • the second fragment was HIS3 dow1 (SEQ IN No. 19)
  • the third fragment was KmURA3 (SEQ IN). No. 20)
  • the fourth paragraph is HIS3 dow2 (SEQ IN No. 21).
  • HIS3 was knocked out from the K. cerevisiae FIM-1 genome by homologous recombination method.
  • the pMD-18T his3 ⁇ -cas plasmid was digested with BamH1 and Kpn1 at 33.5 °C for 3 h, and the 3206 bp fragment was recovered after digestion to obtain his3 ⁇ cassette. This fragment was then transformed into a FIM-1 (ura3 ⁇ ) strain.
  • the Km HIS3 homologous fragment at the 5' and 3' ends and the Km HIS3 gene on the genome are homologously recombined under the screening pressure, and the mutant DNA fragment is replaced.
  • Original genome Km HIS3 gene position Using the Km URA3 tag gene, positive colonies were screened under Uracil-deficient culture conditions, and the identified strains contained the Km URA3 tag, which can normally express the Km URA3 gene-encoded protein.
  • the positive strain was cultured under the condition of 5-FOA drug culture, and the endogenous homologous recombination of the 565 bp homologous fragment of Km HIS3 downstream of Km URA3 on the transforming DNA fragment was carried out by the screening pressure of the drug, and Km URA3 The label is removed from the genome so that the Km URA3 tag can be reused.
  • the transformed bacterial liquid was applied to SC-URA plates, and cultured at 30 ° C for 48-72 hours, and then monoclonal antibodies were applied to SC-Uracil and SC-Histidine plates, respectively.
  • the clones grown on the SC-Uracil plate and not grown on the SC-Histidine plate were transformed into positive clones, which are the his3 ⁇ strain containing the Km URA3 tag gene at the position of the genomic Km HIS3.
  • the strain was applied to YEPD+5-FOA plate, and the inverted clone was cultured at 30 °C for 12-16 hours.
  • the positive clone was endogenously homologously recombined, and the Fm-2 (ura3 ⁇ his3 ⁇ ) strain with the Km URA3 tag gene was dropped.
  • the FIM-2 (ura3 ⁇ his3 ⁇ ) strains identified by sequencing were coated with SC-Uracil and SC-Histidine plates, and could not grow after 12-16 hours of inversion culture at 30 °C, as shown in the last two photos of Figure 10.
  • primers KmAED2-F SQE ID No.27
  • KmAED2-R SQE ID No.28
  • primers KmAED2-F SQE ID No.27
  • KmAED2-R SQE ID No.28
  • the KmAED2 sequence SEQ IN No. 26
  • the sequencing results were aligned with the K.latics KLLA0E02685g gene sequence with a homology of 86.8%.
  • the amino acid sequence of the translated protein sequence of the DNA sequence SEQ IN No. 25
  • the K. latics KLLA0E02685g protein have an amino acid sequence of 87%.
  • primers were designed firstly in the upstream and downstream, and 722bp DNA fragment (ADE2 up) upstream of KmADE2 sequence, 617bp DNA fragment (ADE2 dow1) and 693bp DNA fragment (ADE2 dow2) downstream of KmADE2 sequence were obtained by PCR amplification.
  • Km URA3 1430 bp tag gene sequence SEQ ID No. 22: Chromosome 1:1542629-1544057, 1430 bp
  • the pMD-18T empty plasmid was double-digested with Pstl and Smal, and the obtained linearized vector was recovered.
  • the pMD-18T ade2 ⁇ -cas plasmid was digested with Sph1 and Kpn1 at 37 ° C for 3 h, and the 3462 bp fragment was recovered after digestion.
  • This DNA fragment was transformed into a FIM-1 (ura3 ⁇ ) strain to construct a FIM-4 (ura3 ⁇ ade2 ⁇ ) engineered strain; the DNA fragment was transformed into a FIM-2 (ura3 ⁇ his3 ⁇ ) strain, and a FIM-5 (ura3 ⁇ his3 ⁇ ade2 ⁇ ) engineering strain was constructed.
  • the transformed bacterial solution was coated on SC-Uracil plate, and cultured at 30 ° C for 48-72 hours, and then monoclonal antibodies were applied to SC-Uracil and SC-Adenine plates, respectively.
  • the clones grown on SC-Uracil plates and not growing on SC-Adenine plates were transformed positive clones, which were the ade2 ⁇ strains containing the Km URA3 tag gene at the position of the genome Km ADE2.
  • the strain was applied to YEPD+5-FOA plate, and after 12-16 hours of inversion culture at 30 °C, endogenous homologous recombination occurred in the positive clone, and the Km URA3 tag gene was dropped.
  • YEPD+5-FOA plate positive clones were picked in YEPD liquid medium and cultured at 30 °C for 24 to 48 hours. The genome was extracted and the ade2 ⁇ fragment was amplified by PCR and sequenced. After sequencing, the ade2 ⁇ sequence and the Km ADE2 sequence were subjected to alignment analysis, and the FIM-4 (ura3 ⁇ ade2 ⁇ ) strain and the FIM-5 (ura3 ⁇ his3 ⁇ ade2 ⁇ ) strain ade2 ⁇ sequence were deficient by 120 bp compared to the Km HIS3 sequence.
  • the FIM-4 (ura3 ⁇ ade2 ⁇ ) strains identified by sequencing were coated with SC-Uracil and SC-Adenine plates, and were unable to grow after 12-16 hours of inversion culture at 30 °C (Fig. 12).
  • the FIM-5 (ura3 ⁇ his3 ⁇ ade2 ⁇ ) strains identified by sequencing were coated with SC-Uracil, SC-Histidine, and SC-Adenine plates, and were unable to grow after 12-16 hours of inversion culture at 30 °C (Fig. 13).
  • the K.marxianus URA3 gene sequence was obtained by NCBI database search, and the primers KmURA3-F and KmURA3-R were designed based on the obtained URA3 gene sequence.
  • a KmURA3 DNA fragment containing 3123 bp (Chromosome1:1541868-1544992, 3123 bp) containing the URA3 ORF was obtained by PCR amplification.
  • the above KmURA3 DNA fragment was transformed into FIM-2 (ura3 ⁇ his3 ⁇ ) strain and FIM-5 (ura3 ⁇ his3 ⁇ ade2 ⁇ ) strain by LiOAc chemical transformation, and URA3 was complemented by homologous recombination, and FIM-3 (his3 ⁇ ) and FIM-6 (his3 ⁇ ade2 ⁇ ) were screened. Engineering strains.
  • the FIM-3 (his3 ⁇ ) strain was coated with SC-Uracil plates, and grown in inverted culture at 12 ° C for 12-16 h, while SC-Histidine plates were coated and could not grow after inverted culture at 30 ° C for 12-16 h, as shown in FIG.
  • the FIM-6 (his3 ⁇ ade2 ⁇ ) strain was coated with SC-Uracil plates, and grown in inverted culture at 12 ° C for 12-16 h, while SC-Histidine and SC-Adenine plates were coated, and the cells were unable to grow after 12-16 h of inversion culture at 30 ° C.
  • the Kluyveromyces genome was amplified with primer F1 (SEQ ID No. 79) and primer R1 (SEQ ID No. 80), which was designated as an A fragment containing a sequence of 854 bp to 131 bp upstream of the inulinase gene.
  • the Kluyveromyces genome was amplified with primer F2 (SEQ ID No. 81) and primer R2 (SEQ ID No. 82), which was designated as a B fragment containing a sequence of 1 bp to 850 bp downstream of the inulinase gene.
  • the Kluyveromyces genome was amplified with primer F3 (SEQ ID No. 83) and primer R3 (SEQ ID No. 84), which was designated as a C fragment containing the promoter and ORF of the URA3 gene.
  • the Kluyveromyces genome was amplified with primer F4 (SEQ ID No. 85) and primer R4 (SEQ ID No. 86), which was designated as a D fragment containing a sequence of 331 bp to 1029 bp of the inulinase gene.
  • E Fragments A, B, C, and D are joined, and the joined fragments are named E.
  • the E fragment was transferred into a strain of Kluyveromyces marxianus mutated in the URA3 gene, and transformants were selected on SC medium lacking Uracil. Positive transformants were plated on YPD plates containing 5-Fluoroorotic Acid (5FOA, 2 g/L) to obtain back-screened clones.
  • the genome of the reverse-screened clone was amplified using primers F5 (SEQ ID No. 87) and R5 (SEQ ID No. 88), and the 2300 bp product obtained was a Kluyveromyces strain that knocked out the inulinase gene.
  • Example 6 for use in an auxotrophic strain of K. marxianus CGMCC No. 10621. Recombinant vector for secretion and expression of foreign genes
  • the construction method of the recombinant vector PUKD112 in this embodiment is as follows:
  • Step 1 Amplify the pUC19 plasmid
  • Amplification of pUC19 using primers Plasmid.
  • the PCR product was recovered and the product was designated as the A fragment.
  • Step 2 augment the pcYGW of the Gateway system carrier
  • the pcYGW of the Gateway system vector was amplified using the primer (forward primer SEQ ID No. 49, reverse primer SEQ ID No. 50).
  • the PCR product was recovered and the product was designated as a B fragment.
  • Step 3 amplifying the PKD1 vector
  • the PKD1 vector was amplified using the primer (forward primer SEQ ID No. 51, reverse primer SEQ ID No. 52).
  • the PCR product was recovered and the product was designated as a C fragment.
  • Step 4 connect segments A, B, C
  • Step 5 amplify the PUC19-PKD1 plasmid
  • the PUC19-PKD1 plasmid was amplified using the primer (forward primer SEQ ID No. 53, reverse primer SEQ ID No. 54).
  • the PCR product was recovered and the product was designated as a D fragment.
  • a partial PUC19 sequence and a PKD1 sequence are included in the D fragment.
  • the genome of K. marxianus was amplified using primers (forward primer SEQ ID No. 55, reverse primer SEQ ID No. 56) according to the instructions of the Yeast Genome Extraction Kit (DP307).
  • the PCR product was recovered and the product was named E fragment.
  • the E fragment contains the promoter and ORF of the URA3 gene.
  • Step 7 Amplify the promoter and signal peptide fragment of the inulinase gene
  • the genome of K. marxianus was amplified using primers (forward primer SEQ ID No. 57, reverse primer SEQ ID No. 58).
  • the PCR product was recovered and the product was designated as an F fragment.
  • the F fragment contains a promoter of the inulinase gene, a signal peptide, and a partial multiple cloning site.
  • Step 8 amplifying the terminator fragment of the inulinase gene
  • the genome of K. marxianus was amplified using the primer (forward primer SEQ ID No. 59, reverse primer SEQ ID No. 60).
  • the PCR amplification conditions were the same as in step 1, except that the extension time was 1 minute.
  • the PCR product was recovered according to the instructions of Simgen's gel recovery kit and the product was named G fragment.
  • the G fragment contains a terminator and a partial multiple cloning site of the inulinase gene.
  • Step 9 connect the D, E, F, and G segments
  • the fragments D, E, F, and G were ligated and transformed into Escherichia coli, and the obtained plasmid was named PUKDN112.
  • PUKDN112 was sequenced by Jie Li Company. Referring to Figure 15, the sequencing results are as follows:
  • beta-lactamase ammonium resistance gene
  • 9173-9192 Multiple cloning sites, including three sites: Smal/Xmal, Spel, and Notl.
  • the foreign gene was inserted into the multiple cloning site of PUKDN112, and the sequence of the gene was determined by sequencing to be consistent with the reading frame of the inulinase signal peptide.
  • the transformation was carried out according to the reference using the LiAc method (World Journal of Microbiology & Biotechnology 16:653-654, 2000).
  • the PUKDN112 plasmid containing the foreign gene was transferred into the URA3 gene mutant K. marxianus CGMCC No. 10621 strain, and the URA3 + transformant was screened on the medium lacking Uracil.
  • URA3 + transformants can be used for secretory expression of foreign genes, and methods for expression and detection are performed according to references (Appl Microbiol Biotechnol (2005) 67: 364-369).
  • the PPIC9K vector was amplified with a primer (forward primer SEQ ID No. 90, reverse primer SEQ ID No. 91).
  • the PCR product was recovered and the product was designated as the A fragment.
  • the S fragment contains the Saccharomyces cerevisiae alpha factor signal peptide.
  • the A fragment was probed against the PUKDN112 plasmid according to the instructions of the Agilent QuikChange II kit.
  • the mutated plasmid was PUKDN117. Sequencing was performed by Jie Li Company to determine the sequence of the alpha signal peptide.
  • the PUKDN112 plasmid was mutated according to the instructions of the Agilent QuikChange II kit, and the primers used for the mutation were SEQ ID No. 65 and SEQ ID No. 66.
  • the mutated plasmid was PUKDN115. Sequencing was performed by Jie Li Company to determine the sequence of the multiple cloning site. Referring to Figure 17, the sequencing results are as follows:
  • 9095-9114 Multiple cloning sites, including Spel, Smal/Xmal, and Notl.
  • Step 1 The PUKDN112 vector was amplified with the primer (forward primer SEQ ID No. 67, reverse primer SEQ ID No. 68). The PCR product was recovered and the product was designated as the A fragment.
  • the A fragment contains the inulinase promoter, the multiple cloning site, the inulinase terminator, the URA3 promoter and the URA3 ORF.
  • Step 2 The PUKDN112 vector was amplified with the primer (forward primer SEQ ID No. 69, reverse primer SEQ ID No. 70). The PCR product was recovered and the product was designated as a B fragment. B fragment Contains the PKD1 sequence.
  • Fragments A and B were ligated according to the instructions of the NEB Gibson Assembly Master Mix, and the ligated product was transformed into a strain of Kluyveromyces marxianus mutated in the URA3 gene, and transformants were selected on a medium lacking uracil.
  • the genome of the URA3 + transformant was extracted according to the instructions of the Tiangen Yeast Genome Extraction Kit (DP307).
  • the genome was amplified with the forward primer (SEQ ID No. 71) and the reverse primer (SEQ ID No. 72).
  • the PCR amplification conditions were consistent with the conditions of step 1 in the PUKD112 plasmid construction except that the extension time was 1 minute.
  • the PCR product was recovered according to the instructions of Simgen's gel recovery kit and the product was named C fragment.
  • the fragment contains the linking portion of the A fragment and the B fragment. Fragment C was sequenced by Jie Li Company to verify whether the A fragment and the B fragment were correctly connected. Referring to Figure 18, the sequencing results are as follows:
  • 6062-6081 Multiple cloning sites, including three sites: Smal/Xmal, Spel, and Notl.
  • the transformant containing the correct ligation product was subjected to yeast plasmid extraction according to the instructions of Zymoreprep's Zymoprep Yeast Plasmid Miniprep II kit, and the plasmid was obtained as PUKDN118.
  • the PUKD118 plasmid was amplified with the forward primer SEQ ID No. 73 and the reverse primer SEQ ID No. 74.
  • the obtained PCR product was named as a D fragment.
  • the sequence of SEQ ID No. 75 was added to the 5' end of the forward primer for amplifying the exogenous gene ORF, and the sequence of SEQ ID No. 76 was added to the 5' end of the reverse primer for amplifying the exogenous gene ORF. Using the pair of primers Amplify the foreign gene. The obtained PCR product was named as an E fragment.
  • Fragments D and E were ligated according to the instructions of the NEB Gibson Assembly Master Mix, and the ligation product was transformed into a strain of Kluyveromyces marxianus mutated in the URA3 gene, and transformants were selected on a medium lacking uracil.
  • Yeast plasmid extraction of URA3 + transformants was carried out according to the instructions of Zymoreprep's Zymoprep Yeast Plasmid Miniprep II kit.
  • the plasmid was amplified with the forward primer Fp (SEQ ID No. 77) and the reverse primer Rp (SEQ ID No. 78).
  • the PCR product was recovered according to Simgen's gel recovery kit and the product was designated as the F fragment. Fragment F was sequenced by Jie Li Company to verify whether the foreign gene was correctly linked to PUKD118.
  • a transformant containing PUKD118 in which a foreign gene is correctly ligated is selected for secretion expression of a foreign gene, and expression and detection methods are carried out in accordance with references (Appl Microbiol Biotechnol (2005) 67: 364-369).
  • the PUKDN112 plasmid was mutated by mutating PUKDN112 according to the instructions of the Agilent QuikChange II kit.
  • the primers used for the mutation were SEQ ID No. 61 and SEQ ID No. 62.
  • the mutated plasmid is PUKDN114. Sequencing was performed by Jie Li Company to determine the hexa-histidine tag (6His Tag) sequence and the multiple cloning site. Referring to Figure 19, the sequencing results are as follows:
  • beta-lactamase ammonium resistance gene
  • 9116-9135 Multiple cloning sites, including Spel, Smal/Xmal, and Notl.
  • the PUKDN112 plasmid was mutated.
  • the primers used for the mutation were SEQ ID No. 63 and SEQ ID No. 64.
  • the mutated plasmid is PUKDN116.
  • Sequencing by Jie Li Company confirmed the 6 His Tag sequence and the multiple cloning site. Referring to Figure 20, the sequencing results are as follows:
  • 9095-9114 Multiple cloning sites, including Spel, Smal/Xmal, and Notl.

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Abstract

一种马克斯克鲁维酵母菌，尤其是保藏号为CGMCC No.10621的马克斯克鲁维酵母菌，所述马克斯克鲁维酵母菌的应用，包括构建重组蛋白表达系统、发酵，用于所述马克斯克鲁维酵母的表达载体、所述酵母菌表达的酶/蛋白，以及上述任意物质在饲料、饲料添加剂、食品、疫苗、药物等领域中的应用。马克斯克鲁维酵母菌具有高的分泌蛋白的能力和高的生物量，可用于生产酶制剂，并且可以用来构建重组蛋白表达系统，并且，马克斯克鲁维酵母菌能提供蛋白、核酸、多糖、维生素等营养成分，补充动物生长过程中所需营养成分，能帮助动物抗氧化、代谢乳糖及脂质。

Description

马克斯克鲁维酵母菌及其应用 技术领域
本发明涉及一种酵母菌，尤其涉及一种具有高分泌性能和高生物量的马克斯克鲁维酵母菌、马克斯克鲁维酵母表达系统、及其应用，本发明还涉及所述酵母菌表达的酶/蛋白，以及上述物质在饲料、饲料添加剂、食品、疫苗、药物等领域中的应用。
背景技术
马克斯克鲁维酵母(Kluyveromyces marxianus，K.marxianus)在自然界中普遍存在于酸马奶(Khrisanfova L P，Antimicrobial properties of Koumiss from cow and mare milk，Moloch.Prom.，1969，30(10)：16)、酸奶(Fleet GH，Spoilage yeasts，Crit Rev Biotechnol.，1992，12：1-44；Tudor A等人，Food spoilage yeasts，In Rose AH，Harrison JS(eds)The yeasts，vol 5，2nd edn.Academic Press，London，1993：435-515.)、牛奶(Wendel B.Silveira等人，Genomic Sequence of the Yeast Kluyveromyces marxianus CCT 7735(UFV-3)，a Highly Lactose-Fermenting Yeast Isolated from the Brazilian Dairy Industry.Genome Announcements，2014，2(6)：e01136-14)、水果(Lodder J等人，The yeasts：a taxonomic study，NHPC，Amsterdam，1952)、甘蔗叶子(Lodder J等人，The yeasts：a taxonomic study，NHPC，Amsterdam，1952)等安全环境中；马克思克鲁维酵母(Kluyveromyces marxianus)已经通过了美国和欧洲的GRAS和QPS安全认证，其自身不仅被认为是安全的微生物菌株，同时也被认为是可用于制备食品级重组蛋白(酶)的安全微生物菌株，是一种被欧盟认可的食品级的酵母。它具有营养要求极其简单、生长旺盛、生物量大、生长温度适应范围广等特点。同时，它还具有高效分泌蛋白的特点。因此，马克思克鲁维酵母具有高效表达食用和饲用蛋白的巨大潜力。
发明内容
本申请提供了一种具有高分泌性能和高生物量的马克斯克鲁维酵母菌、马克 斯克鲁维酵母表达系统、所述酵母菌表达的酶/蛋白，以及在饲料、饲料添加剂、食品、疫苗、药物等领域中的应用。
本发明第一个方面是提供一种马克斯克鲁维酵母菌(Kluyveromyces marxianus；K.marxianus)，该马克思克鲁维酵母菌具有SEQ ID No.1所述基因序列、或与SEQ ID No.1同源度至少为90％的基因序列。
在一种优选实施例中，所述马克斯克鲁维酵母菌具有与SEQ ID No.1同源度为至少93％、优选为至少95％、更优选为至少98％、更优选为至少99％的基因序列。
在一种优选实施例中，所述马克斯克鲁维酵母菌保藏号为CGMCC No.10621(在本发明下述内容中，也可以称为马克斯克鲁维酵母FIM-1、K.marxianus FIM-1或Kluyveromyces marxianus FIM-1)。
本发明所述的马克斯克鲁维酵母菌CGMCC No.10621，保藏于中国微生物菌种保藏管理委员会普通微生物中心；保藏单位地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所；保藏日期为2015年3月13日。
本发明马克斯克鲁维酵母菌CGMCC No.10621，株菌落为圆形，呈乳白色，边缘整齐；酵母菌呈椭圆形，大小约为4-8μm，出芽生殖，单个、两个或多个系成串粘连。
本发明马克斯克鲁维酵母菌，发酵分泌内切菊粉酶，并且在以葡萄糖为碳源条件下菊粉酶产量大于葡萄糖、菊粉混合碳源条件下的产量。
本发明马克斯克鲁维酵母菌，菌株接种量0.05％-5％，按照碳氮比C∶N＝20∶1-5∶1的比例，25-38℃发酵36h时细胞生物量≥100g/L。
本发明第二个方面是提供一种马克斯克鲁维酵母菌构建的酵母表达系统，优选为外源蛋白的重组表达系统。所述酵母表达系统、优选为外源蛋白的重组表达系统可以是所述马克斯克鲁维酵母的基因突变株、基因工程手段获得的菌株中的任意一种或几种，并且，所述基因突变株、基因工程手段获得的菌株具有与本发明第一个方面所述马克斯克鲁维酵母菌相似的生物量。
其中，所述“相似”是指与本发明第一个方面所述马克斯克鲁维酵母菌之间生物量数据差异≤5％，更优选为≤3％。
其中所述马克斯克鲁维酵母优选为本发明第一个方面所述的马克斯克鲁维 酵母菌，更优选为马克斯克鲁维酵母CGMCC No.10621。
本发明提供了一种马克斯克鲁维酵母的营养缺陷型菌株，即敲除全部或部分营养基因之后的菌株。
其中，所述营养基因优选为选自URA3基因、HIS3基因、ADE2基因中的任意一种或几种。
其中，在一种优选实施例中，所述马克斯克鲁维酵母菌株敲除全部或部分URA3基因。
其中，所述敲除部分基因URA3基因优选为至少敲除该基因全部或部分ORF区，更优选为至少敲除该基因全部或部分CDS区。
其中，在一种更优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株在Uracil营养缺陷培养基中不能生长。
本发明所述的马克斯克鲁维酵母营养缺陷菌株，还可以是所述马克斯克鲁维酵母菌株敲除全部或部分HIS3基因、ADE2基因中的任意一种或几种之后的菌株。
其中，更优选地，敲除全部或部分HIS3基因后，所述马克斯克鲁维酵母营养缺陷菌株在SC-Histidine营养缺陷培养基中不能生长。
其中，更优选地，敲除全部或部分ADE2基因后，所述马克斯克鲁维酵母营养缺陷菌株在SC-Adenine营养缺陷培养基中不能生长。
在一种优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株，是马克斯克鲁维酵母菌株敲除全部或部分URA3基因、以及全部或部分HIS3基因的菌株。
在一种更优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株在SC-Uracil、SC-Histidine营养缺陷培养基中不能生长。
在一种优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株，是马克斯克鲁维酵母菌株敲除全部或部分URA3基因、以及全部或部分ADE2基因的菌株。
在一种更优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株在SC-Uracil、SC-Adenine营养缺陷培养基中不能生长。
在一种优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株，是马克斯克鲁维酵母菌株敲除全部或部分URA3基因、全部或部分HIS3基因、以及全部或部分ADE2基因的菌株。
在一种更优选实施例中，所述的马克斯克鲁维酵母营养缺陷菌株在SC-Uracil、SC-Histidine、SC-Adenine营养缺陷培养基中不能生长。
在一种优选实施例中，所述马克斯克鲁维酵母营养缺陷菌株，是马克斯克鲁维酵母菌株敲除全部或部分HIS3基因之后的菌株。
其中，在一种优选实施例中，所述敲除部分HIS3基因为至少敲除全部或部分该基因的ORF区，更优选为至少敲除全部或部分该基因的CDS区。
其中，在一种优选实施例中，所述的马克斯克鲁维酵母营养缺陷菌株在SC-Histidine营养缺陷培养基中不能生长。
在一种优选实施例中，所述的马克斯克鲁维酵母营养缺陷菌株，是马克斯克鲁维酵母菌株敲除全部或部分HIS3、以及全部或部分ADE2基因的菌株。
在一种更优选实施例中，所述的马克斯克鲁维酵母营养缺陷菌株在SC-Histidine、SC-Adenine营养缺陷培养基中不能生长。
本发明还提供一种马克斯克鲁维酵母菌构建的酵母表达系统，更优选为马克斯克鲁维酵母敲除菊粉酶基因的菌株；或者上述内容中所述马克斯克鲁维酵母营养缺陷型菌株敲除菊粉酶基因的菌株。
其中，所述马克斯克鲁维酵母菌优选为本发明第一个方面所述的马克斯克鲁维酵母菌，更优选为马克思克鲁维酵母CGMCC No.10621。
本发明第三个方面是提供一种用于在马克思克鲁维酵母营养缺陷型菌株中进行外源基因分泌表达的重组载体。其中，所述马克斯克鲁维酵母菌优选为本发明第一个方面所述的马克斯克鲁维酵母菌，更优选为马克思克鲁维酵母CGMCC No.10621。
在本发明第三个方面一种优选实施例中，所述重组载体按照顺序包括PKD1载体、菊粉酶启动子、信号肽、多克隆位点、菊粉酶终止子、营养基因启动子、营养基因开放阅读框(ORF)。
在本发明第三个方面一种优选实施例中，所述重组载体按照顺序包括PKD1载体、菊粉酶启动子、多克隆位点、菊粉酶终止子、营养基因启动子、营养基因开放阅读框(ORF)。
在本发明第三个方面一种优选实施例中，所述重组载体按照顺序包括PKD1载体序列、菊粉酶启动子、多克隆位点、菊粉酶终止子、营养基因启动子、营养 基因开放阅读框(ORF)；其中，所述重组载体还包括多聚组氨酸标签，所述多聚组氨酸标签位于多克隆位点的C端或N端。
其中，本发明第三个方面所述的重组载体还可以包括或不包括氨苄抗性基因；优选地，如果存在氨苄抗性基因，按照顺序，所述PKD1位于氨苄抗性基因与菊粉酶启动子之间。更优选地，所述重组载体不包括氨苄抗性基因。
其中，所述信号肽优选为菊粉酶信号肽、或酵母alpha因子信号肽中的任意一种或几种，并优选为菊粉酶信号肽。在一种更优选实施例中，所述重组载体按照顺序包括PKD1载体、菊粉酶启动子、菊粉酶信号肽、多克隆位点、菊粉酶终止子、营养基因启动子、营养基因开放阅读框(ORF)。
其中，所述多聚组氨酸标签优选四聚或以上组氨酸标签，更优选为五聚或以上组氨酸标签，更优选为六聚或以上组氨酸标。
其中，所述营养基因优选为马克斯克鲁维菌株的营养基因，如URA3基因、HIS3基因、ADE2基因中的任意一种或几种，更优选为URA3基因。
其中，所述多克隆位点可以是包括一个或多个位点限制性酶切位点的序列，所述限制性酶切位点如Smal、Xmal、Spel、Notl中的任意一种或几种，更优选为Spel、Notl、以及Smal和/或Xmal中的任意一种或几种，如Smal/Xmal、Spel、Notl。
其中，所述多克隆位点长度优选为15-25bp，更优选为18-23bp，更优选为19-21bp。
在本发明的一种优选实施例中，所述重组载体的DNA序列选自SEQ ID No.41-SEQ ID No.46、SEQ ID No.89中的任意一种或几种。
本发明还提供了一种转化子，将本发明第三方面所述任意一种或几种重组载体转入马克斯克鲁维酵母的菌株中，优选为转入本发明上述任意一种基因缺陷型马克斯克鲁维酵母的菌株中，更优选为转入URA3基因缺陷型马克斯克鲁维酵母的菌株中。其中，所述马克斯克鲁维酵母菌优选为本发明第一个方面所述的马克斯克鲁维酵母菌，更优选为马克思克鲁维酵母CGMCC No.10621。
本发明第四个方面是提供一种马克斯克鲁维酵母菌在产品发酵或产品添加剂中的应用，其中，所述产品可以是动物饲料、动物饲料添加剂、食品、疫苗、药物中的任意一种或几种，并优选为用作发酵菌剂，如动物饲料添加剂和/或动 物饲料发酵菌剂。
本发明所述马克斯克鲁维酵母菌在饲料发酵或饲料添加剂中的应用能够提高动物免疫力，和/或提高动物消化能力，和/或保护动物不受自由基损伤，和/或生产更多营养成分；更优选为，相比于现有酵母具有更优的提高动物免疫力、和/或提高动物消化能力、和/或保护动物不受自由基损伤、和/或生产更多营养成分的能力。
其中，所述马克斯克鲁维酵母菌优选为本发明第一个方面所述的马克斯克鲁维酵母菌、和/或第二个方面所述的马克斯克鲁维酵母菌构建的酵母表达系统、和/或本发明第三个方面所述的转化子。
本发明所述马克斯克鲁维酵母用作动物饲料添加剂和/或动物饲料发酵菌剂，相比于现有马克斯克鲁维酵母，能够使饲料具有更优的提高动物免疫力、和/或提高动物消化能力、和/或保护动物不受自由基损伤、和/或生产更多营养成分的能力。
其中，更优选实施例中，A)所述发酵菌剂直接添加至动物饲料添加剂和/或动物饲料、食品、疫苗或药物中，或者B)可以是所述发酵菌剂对原料进行发酵制备含有发酵产物的动物饲料添加剂和/或动物饲料、食品、疫苗或药物，或者C)所述发酵菌剂表达的含有酶或蛋白的发酵物添加至动物饲料添加剂和/或动物饲料、食品、疫苗或药物中。
在一种优选实施例中，本发明所述马克斯克鲁维酵母用作动物饲料添加剂和/或动物饲料发酵菌剂，能够提高动物免疫力，如：促进T细胞功能、促进巨噬细胞功能、和提供更强的糖链免疫促进功能。
在一种优选实施例中，本发明所述马克斯克鲁维酵母用作动物饲料添加剂和/或动物饲料发酵菌剂，能够使动物避免或降低D-乳酸中毒，如：能够降低或消除D-乳酸。
在一种优选实施例中，本发明所述马克斯克鲁维酵母用作动物饲料添加剂和/或动物饲料发酵菌剂，能够降低或消除乳糖不耐症。
在一种优选实施例中，本发明所述马克斯克鲁维酵母用作动物饲料添加剂和/或动物饲料发酵菌剂，能够降解脂质，降低或消除脂质在动物消化系统内堆积。
在一种优选实施例中，本发明所述马克斯克鲁维酵母用作动物饲料添加剂和 /或动物饲料发酵菌剂，能够提供消除自由基能力，保护动物不受或少受自由基损伤。如，可以降低动物体内过氧化物。
在一种优选实施例中，本发明所述马克斯克鲁维酵母用作动物饲料添加剂和/或动物饲料发酵菌剂，能够为动物提供生物素和/或蛋白质。
本发明还提供了一种添加剂，可以是动物饲料、食品、疫苗、药物添加剂终端任意一种或几种。所述添加剂至少包括A)本发明第一个方面所述的马克斯克鲁维酵母菌及其表达酶/蛋白、和/或B)第二个方面所述的马克斯克鲁维酵母菌构建的酵母表达系统及其表达酶/蛋白、和/或C)本发明第三个方面所述的转化子及其表达酶/蛋白中的任意一种或几种。
本发明还提供了一种产品，所述产品可以是动物饲料、疫苗、药物、食品中的任意一种或几种，包括至少一种添加剂，所述至少一种添加剂包括A)本发明第一个方面所述的马克斯克鲁维酵母菌及其表达酶/蛋白、和/或B)第二个方面所述的马克斯克鲁维酵母菌构建的酵母表达系统及其表达酶/蛋白、和/或C)本发明第三个方面所述的转化子及其表达酶/蛋白中的任意一种或几种。
本发明还提供了一种发酵菌剂，所述动物饲料发酵菌剂至少包括本发明第一个方面所述的马克斯克鲁维酵母菌、和/或第二个方面所述的马克斯克鲁维酵母菌构建的酵母表达系统、和/或本发明第三个方面所述的转化子。
本发明还提供了一种发酵的产品，可以是选自动物饲料、食品、疫苗或药物中的任意一种或几种，所述发酵的产品是原料在发酵菌剂存在下发酵得到，其中，所述发酵菌剂至少包括本发明第一个方面所述的马克斯克鲁维酵母菌、和/或第二个方面所述的马克斯克鲁维酵母菌构建的酵母表达系统、和/或本发明第三个方面所述的转化子。
其中，本发明上述的任意一种产品、和/或产品添加剂还可以包括营养元素、调味剂、其他有益菌、防霉剂、胆汁酸、螯合肽、复合酶、促生长组分、抗生素、色素、以及其他可用于产品或产品添加剂的组分中的任意一种或几种。
其中，所述发酵菌剂还可以包括其他可用于发酵的酵母。
其中，本发明上述的疫苗、药物可以是用于人类、也可以是用于动物的疫苗、药物。
其中，本发明所述动物可以是优选为如下动物中的任意一种或几种：A)棘 皮动物门、节肢动物们、软体动物门等无脊椎动物；B)鱼纲、两栖纲、爬行纲、鸟纲和哺乳纲等脊椎动物。具体如田螺、蜗牛、鲍鱼、贝类、虾、蟹、海参、海胆、鱼、蛙、蛇、龟、猪、牛、羊、马、狗、猫、鼠、兔、貂、鼬、海豚、鲨鱼、鲸鱼、鸡、鸭、鹅、鹌鹑、鸽、鸵鸟等肉用型动物、蛋用型动物、药用型动物、毛皮用动物、观赏型动物、以及其他经济型动物。
本领域技术人员应当理解的是，在没有特别说明的情况下，本发明所述马克斯克鲁维酵母菌可以是优选为本发明第一个方面所述的马克斯克鲁维酵母菌，更优选为马克思克鲁维酵母CGMCC No.10621。
本发明所提供的马克斯克鲁维酵母菌，相比于ATCC8585、ATCC26548等现有克鲁维酵母，具有更高的分泌蛋白的能力和更高的生物量，可用于生产酶制剂，并且可以用来构建重组蛋白表达系统。
本发明所述的营养缺陷型马克斯克鲁维酵母菌株，可以用于马克斯克鲁维酵母表达系统的宿主菌，用于重组制备外源蛋白(酶)。
并且，发明人出人意料地发现：马克斯克鲁维酵母作为饲料添加剂，比酿酒酵母更能提高动物免疫力，能提供更多的蛋白及生物素，且更能对D-乳酸进行解毒；同时也出人意料地发现本发明马克斯克鲁维酵母、尤其是马克斯克鲁维酵母CGMCC No.10621菌株与其它5株已报道的马克斯克鲁维酵母相比，能提供更多的蛋白，更能帮助动物抗氧化、代谢乳糖及脂质，并且在作为分泌外源蛋白的表达系统方面也有优势。
附图说明
图1为本发明马克斯克鲁维酵母菌分泌蛋白能力，泳道1：本发明菌株K.marxianus FIM-1；泳道2：K.marxianus ATCC 26548；泳道3：K.lactis ATCC8585；泳道M：蛋白Marker；
图2为本发明马克斯克鲁维酵母菌细胞形态；
图3为本发明马克斯克鲁维酵母菌高生物量发酵结果；
图4为马克斯克鲁维酵母CGMCC No.10621基因在其它马克斯克鲁维酵母菌株中的缺失情况示意图，左侧框体中显示的是CGMCC No.10621(FIM-1)的相关基因，右侧方格中灰色表示该基因对应的菌株中不存在；其中，图4A为蛋白 生成相关基因，图4B为细胞快速生长相关基因，图4C为其他基因，如抗氧化能力，乳糖代谢，脂质代谢，以及蛋白分泌过程有关基因；其中：
CCT表示K.maxcianus CCT7735、NBRC表示K.maxcianus NBRC1777、KCTC表示K.maxcianus KCTC17555、DMB1表示K.maxcianus DMB1K，DMKU表示K.maxcianus DMKU3-1042；
图5为pMD-18T ura3Δ-cas质粒构建示意图；
图6为同源重组方法敲除FIM基因组上的URA3基因示意图；
图7为所得FIM-1(ura3Δ)与野生型FIM在尿嘧啶营养缺陷性培养基上的生长状态；
图8为pMD-18T his3Δ-ca质粒构建示意图；
图9为FIM-2(ura3Δhis3Δ)菌株构建原理示意图；
图10为FIM-2(ura3Δhis3Δ)菌株构建筛选过程结果；
图11为pMD-18T ade2ΔURA3质粒构建示意图；
图12为FIM-4(ura3Δade2Δ)菌株平板鉴定结果；
图13为FIM-5(ura3Δhis3Δade2Δ)菌株平板鉴定结果；
图14为FIM-3(his3Δ)菌株平板鉴定结果。
图15为本发明实施例中所构建重组载体PUKD112原理图；
图16为本发明实施例2所构建重组载体PUKD117原理图；
图17为本发明实施例中所构建重组载体PUKD115原理图；
图18为本发明实施例中所构建重组载体PUKD118原理图；
图19为本发明实施例中所构建重组载体PUKD114原理图；
图20为本发明实施例中所构建重组载体PUKD116原理图。
具体实施方式
实施例1，菌株的获得
样品采集和酵母分离：
2014年8月从西藏那曲地区采集不同的酸奶样本。
取酸奶样本1g，用50ml的无菌水稀释，然后用无菌水进行10倍、100倍、1000倍和10000倍稀释，取100μl稀释液涂布于酵母固体培养基YNB(无氨基 酵母氮源；Yeast Nitrogen Base without Amino Acids)+1％菊粉平板上，30℃培养3-4天后形成克隆。
筛选
将酸奶样本中分离到的酵母菌以及从ATCC购买两株克鲁维酵母菌株Kluyveromyces lactis(保藏号：ATCC8585)和K.marxianus(保藏号：ATCC26548)，在含1％酵母提取物(Yeast Extract)和2％的葡萄糖液体培养基中，30℃发酵培养120h后收集发酵上清，然后采用蛋白质电泳技术-SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)检测各株酵母分泌蛋白的能力。
其中发现：一株酵母FIM-1具有高分泌蛋白能力(如图1所示)，分泌蛋白的大小约90kDa左右。
菌株鉴定
酵母菌FIM-1的菌种的鉴定采用了18s rDNA方法。
以提取酵母菌FIM-1的基因组为模板，通过18S rDNA通用引物NS1(gtagtcatatgcttgtctc)和NS8(tccgcaggttcacctacgga)，进行PCR扩增与测序，获得了18s rDNA序列如SEQ ID NO.1所示，与GenBank中的已知序列进行比对后，发现获得的酵母菌FIM-1的18s rDNA序列与K.marxianus NBRC1777的序列相似度为100％。
细胞形态学观察发现，酵母菌FIM-1的株菌落为圆形，呈乳白色，边缘整齐，显微下观察发现该酵母菌呈椭圆形(如图2)，大小约为4-8μm，出芽生殖，单个、两个或多个系成串粘连，这些表现与马克斯克鲁维酵母的细胞形态基本一致，因此该酵母菌命名为K.marxianus FIM-1。
本发明所述的马克斯克鲁维酵母菌，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏单位地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏日期为2015年3月13日，保藏号为CGMCC No.10621。
实施例2、高生物量发酵
1)初始培养基：酵母粉40g，葡萄糖50g，初始体积1L。灭菌：115℃，20min。
2)发酵过程控制：pH7.0，用30％氨水调节、和10％HCl调节；溶解氧设定30％DOT，依靠转速调节溶解氧。
3)培养基流加：开始流加培养基时间是6-10小时，残糖浓度约为5-15g/L。
4)发酵培养时间：48-72小时。培养温度：28-35℃。
实验结果：
按照碳氮比C∶N＝20∶1-5∶1的比例，补料流速采用20-35g/h葡萄糖的流速进行碳源和1-1.25g/h氮源补料，发酵周期为48-96小时，放罐时发酵液总体约为2.2-3.5L。发酵过程消耗葡萄糖总量为850-1110克，酵母浸出物消耗量约180-232克，酵母细胞生物量约78-117g/L(干重)，细胞产率为0.56-0.62，细胞最大生长速度为2.92-3.29g/L。其中以20g/L葡萄糖流速进行补料获得的细胞密度及细胞生物量最高，如图3所示，发酵36h时细胞生物量110.1g/L，发酵57h时细胞生物量达117.2g/L(干重)，发酵过程中的细胞产率为0.62。
实施例3，马克斯克鲁维酵母CGMCC No.10621在饲料中的应用
图4是基于15株酵母中都单一存在的基因组成的长序列构建得到。15株酵母如下所示：
Figure PCTCN2016094868-appb-000001
其中，表格C部分内是位于双足囊菌科的Yarrowia lipolytica酵母以及位于法夫酵母科的Pichia pastoris酵母，它们在酵母目下较早分化出，之后独立进化。
表格B部分和A部分内均为酵母科，通常称为“Saccharomyces complex”。其中，表格B部分内的酵母与Saccharomyces属的进化关系较近，常在葡萄酒酿制过程中出现；表格A部分内的酵母与Kluyveromyces属的进化关系更近，其中Kluyveromyces属多位于乳制品中，Eremothecium属多位于棉花种植中。
提高动物自身的免疫力
1)a-1，3-甘露聚糖可以促进T细胞功能
S.cerevisiae酵母细胞壁糖蛋白N-寡聚糖链上的a-1，3连接的甘露糖基，有较高的免疫原性，能够增加细胞因子的释放，提高白介素IL-2浓度以促进T细胞的增殖和分化，且增强干扰素活性，从而提高动物对细菌等疾病的非特异性免疫力。
表1和表2给出了酵母种间及种内关键重复基因个数图，表1为种间15株酵母各组的重复基因个数，表2为K.marxianus(K.M.)种内6株酵母重复基因个数。
通过表1和表2可以看出，编码a-1，3甘露糖基转移酶的基因ALG3在K.marxianus酵母中的拷贝个数，普遍地高于其它酵母物种，这提示K.marxianus细胞壁中可能具有更多的a-1，3连接的甘露糖基，从而促进动物的免疫力，因此，相比于其他酵母，K.marxianus能够具有更佳的提高动物自身免疫力的能力。
2)β-1，3葡聚糖促进巨噬细胞功能
酵母细胞壁中β-葡聚糖的单糖之间以β-1，3键或β-1，6键相连，这种特殊构型对动物免疫系统有较强刺激作用，能直接产生大量巨噬细胞，从而清除体内损伤、衰老的自身细胞及侵入体内的病原微生物。
通过表1和表2可以看出，编码β-1，3葡聚糖基转移酶的基因GAS3在K.marxianus中的拷贝个数比S.cerevisiae更多，从而更能促进巨噬细胞的免疫。
3)N-糖基化修饰
图4给出了K.marxianus CGMCC No.10621基因在其它K.marxianus.菌株中的缺失情况，图中灰色方格代表K.marxianus CGMCC No.10621中的某基因在相应的其它K.marxianus菌株中缺失。图4A部份主要与蛋白生成有关，图 4B部分主要与线粒体产生能量以及K.marxianus细胞快速生长有关，图4C部分与抗氧化能力，乳糖代谢，脂质代谢，以及蛋白分泌过程有关。
参照图4C，K.marxianus CGMCC No.10621中N-糖基化起始步骤寡糖核心转移酶的一个亚基STT3在K.marxianus NBRC1777中缺失(图4C最下方)，这意味着K.marxianus比K.marxianus NBRC1777具有更强的糖链免疫促进功能。
消除有害的D-乳酸
乳酸分成L-乳酸与D-乳酸。人及动物能消耗利用L-乳酸，而但不能代谢D-乳酸，过量摄入易引起代谢紊乱等不良反应。在酸奶及乳酸饮料中，主要是L-乳酸，仅有很少量的D-乳酸。
参照表1和表2，编码D-乳酸脱氢酶的基因DLD(能将D-乳酸转化成丙酮酸)普遍地在Kluyveromyces属中的拷贝数远多于其它酵母物种，这意味着K.marxianus在D-乳酸消耗方面有更大的能力，从而保护动物免于D-乳酸中毒。
降低乳糖不耐症
已有研究报道，有些仔猪存在乳糖不耐受症，即缺乏乳糖酶时，摄入的乳糖滞留在肠道，被肠道有害细菌发酵利用，产生醋酸、丙酸、丁酸等，并产生大量气体包括CH4、H2、CO2，导致腹胀及腹泻。
参照图4C，K.marxianus CGMCC No.10621中参与乳糖利用的一些基因如GAL4和HNT1在现有菌株K.marxianus NBRC1777及DMKU3-1042中有缺失，而在CGMCC No.10621中存在，即：意味着K.marxianus CGMCC No.10621以活性干粉形式添加到饲料中后，在动物肠道中可以代谢乳糖，提高动物特别是仔猪肠道健康。
降解脂质
参照图4，参与脂质降解中脂酰辅酶A运输的基因ACBP在K.marxianus NBRC1777中缺失(图4C右下图所示)，这意味着K.marxianus CGMCC No.10621降解脂质的能力可能更强，以防止食物中过多的脂质在动物肠道堆积而引起腹泻。
表1，酵母种间关键重复基因个数图
Figure PCTCN2016094868-appb-000002
表2，K.marxianus种内关键重复基因个数图
Figure PCTCN2016094868-appb-000003
Figure PCTCN2016094868-appb-000004
较强的抗氧化能力，清除自由基
参照表1、表2和图4，可以看出：
1)参与细胞中NAD再生的基因NQO2在K.marxianus中存在且都有多个拷贝，而在大多数其它酵母物种中该基因都缺失(表1)，这意味着K.marxianus具有更高效地生成NAD的能力；
2)K.marxianus CGMCC No.10621中促进NADP生成的基因PPNK，在菌株K.marxianus DMB1中缺失(图4C部分最上方左图所示)；
3)K.marxianus CGMCC No.10621中参与氧化还原反应平衡以消除过氧化物的一些基因，在其它5株K.marxianus酵母中各有缺失(图4C右边中间部分)。由于辅酶NAD+与NADH参与氧化还原平衡，NADP与NADPH能够为细胞中的谷胱甘肽补充还原力，且其它基因是直接消除过氧化物，因此K.marxianus CGMCC No.10621与其它K.marxianus菌株相比，具有更强的消除自由基能力，即具有更强的保护动物不受自由基损伤的能力。
生产更多的生物素
B族维生素中的生物素，在促进细胞中的羧化反应时起了重要作用，比如线粒体中丙酮酸羧化形成草酰乙酸，从而对TCA循环进行补料。动物不能自身合成生物素，除了食物来源(比如牛奶，草莓，葡萄等)之外，还有部分来源于肠道中的菌落。
通过表1和表2可以看出，参与生物素合成的基因BIOA(编码腺苷甲硫氨酸-8-氨基-7-氧代壬酸化物转氨酶)及BIOD(编码脱硫生物素合酶)，在K.marxianus物种中的拷贝个数明显多于其它酵母物种。这意味着K.marxianus能为动物提供更多的生物素。
生产更多的细胞蛋白
通过表1和表2可以看出，细胞膜上参与尿素转运的基因DUR3以及参与甲硫氨酸转运的基因MUP1&3在K.marxianus中的拷贝数普遍高于其它酵母，特别是明显高于S.cerevisiae，这意味着K.marxianus能够产生更多的单细胞蛋白，从而为动物提供更大量的蛋白质食物来源。
而在K.marxianus物种内部，如图4A部分所示，K.marxianus CGMCC No.10621中一些参与pre-mRNA加工的基因、以及一些核糖体蛋白的基因在菌株NBRC1777及菌株DMKU3-1042中有缺失；K.marxianus CGMCC No.10621中细胞膜上负责把细胞内部高浓度的氨外运的基因YOR378W，在菌株CCT7735及菌株NBRC1777中缺失。由于mRNA的成熟及核糖体的效能，影响了蛋白生成；而把细胞中高浓度的氨运出，也间接说明了细胞内有机氮源充足，为蛋白的合成准备了充分的原料，因此，K.marxianus CGMCC No.10621在蛋白合成的量及效率方面，相对于现有K.marxianus菌株CCT7735、NBRC1777、及DMKU3-1042，均有一定优势。
另外如图4B部分所示，K.marxianus CGMCC No.10621中一些参与线粒体功能及能量生成基因，主要在菌株NBRC1777中有缺失，在菌株KCTC17555中也有少量缺失；且K.marxianus CGMCC No.10621中一些参与染色体分离以及细胞骨架的基因，在菌株K.marxianus NBRC1777中也有缺失。这意味着K.marxianus CGMCC No.10621有更快的生长速率，因此能更快地提供细胞蛋白。
更完善的蛋白分泌过程
参照图4C，K.marxianus CGMCC No.10621中参与蛋白分泌通路的基因(包括参与新生肽链进入ER、剪切信号肽、ER到高尔基体运输、以及分泌的最后胞吐过程)，在现有K.marxianus菌株NBRC1777及DMKU3-1042中都存在缺失，这意味着K.marxianus CGMCC No.10621在蛋白分泌通路上更为完善，即也适用于作为外源蛋白表达系统。
上述实施例证明，马克斯克鲁维酵母CGMCC No.10621酵母具有促进动物免疫力的功能，为动物生长提供丰富的蛋白、生物素，且帮助去掉有害的D-乳酸、乳糖、自由基，可用于动物饲料添加和/或饲料发酵。
实施例4，马克斯克鲁维酵母CGMCC No.10621无痕基因组改造
敲除ura3基因的马克斯克鲁维酵母工程菌株FIM-1（ura3Δ)的构建
1、KmURA3基因克隆和pMD-18T ura3Δ-cas质粒构建
通过NCBI数据库检索获得K.marxianus的URA3基因序列信息，并设计引物URA3F(SQE ID No.2)、URA3R(SQE ID No.3)扩增获得FIM URA3基因序列3123bp。
参照图5，在URA3 CDS(Chromosome1：1543214-1544017，804bp)上下游分别设计引物URA上游F(SQE ID No.4)、URA3上游R(SQE ID No.5)、URA3下游F(SQE ID No.6)、URA3下游R(SQE ID No.7)。
PCR扩增获得URA3 CDS上游930bp DNA片段(Chromosome1：1541868-1542797，930bp)，和下游944bp DNA片段(Chromosome1：1544046-1544992，944bp)。同时，pMD-18T空载质粒用Pstl、Smal双酶切后回收获得的线性化载体片段。将上述三个片段通过一步克隆法连接，获得URA3基因敲除的中断盒pMD-18T ura3Δ-cas。
2、FIM-1(ura3Δ)菌株筛选和验证
参照图6，ura3Δcassette线性DNA片段与马克斯克鲁维酵母CGMCC No.10621基因组序列发生同源重组(homologous recombination)，实现对基因组上ura3的敲除。首先以URA3缺失突变质粒pMD-18T ura3Δ-cas为模板，PCR扩增获得缺失突变片段ura3Δcassette线性DNA。将该片段转化K.marxianus FIM野生菌株，在含有0.2％5-FOA的YEPD平板上30℃培养48h后，即可筛选获得耐5-FOA的阳性菌。
挑取在含有0.2％5-FOA的YEPD平板上生长的单菌落，接种到含有0.2％5-FOA的YEPD液体培养基中培养，并提取基因组DNA。PCR扩增、测序，分析后证实FIM-1的基因组上缺失了URA3 CDS共1249bp(Chromosome1：1542797-1544046，1249bp)，将该缺失突变菌株命名为FIM-1(ura3Δ)。
同时在Uracil营养缺陷培养基上进行培养，发现FIM-1(ura3Δ)不能生长，而野生型的FIM(wt)生长良好，如图8所示。
敲除ura3、his3基因的马克斯克鲁维酵母工程菌株FIM-2(ura3Δhis3Δ)的构建
1、KmHIS3基因克隆和pMD-18T his3Δ-cas质粒构建
通过NCBI检索获得的关于K.marxianus的HIS3基因序列信息，设计引物 (表3)，通过PCR扩增得到HIS3上游和下游各600bp左右的同源序列(HIS3up(607bp)、HIS3 dow1(565bp)、HIS3 dow2(604bp))，以及Km URA31430bp标签基因序列(Chromosome1：1542629-1544057，1430bp)。
表3，扩增HIS3基因所用引物
HIS3 up-F SEQ ID No.8
HIS3 up-R SEQ ID No.9
HIS3 dow2-F SEQ ID No.10
HIS3 dow2-R SEQ ID No.11
KmURA3-F SEQ ID No.12
KmURA3-R SEQ ID No.13
HIS3 dow1-F SEQ ID No.14
HIS3 dow1-R SEQ ID No.15
HIS3 dow1-KmURA3-F SEQ ID No.16
HIS3 dow1-KmURA3-R SEQ ID No.17
参照图8，同时将pMD-18T空载质粒用Pstl、Smal双酶切后回收获得的线性化载体。将上述各DNA片段通过一步克隆法先后3次将4段DNA片段连到载体上，即获得pMD-18T his3Δ-cas质粒。
参照图9，首先将HIS3 up和HIS3 dow2两个DNA片段利用其10-25bp同源序列，通过一步克隆法连接在pMD-18T线性化载体上；利用之前在HIS3 up和HIS3 dow2同源序列末端添加的Smal酶切位点单酶切线获得包含HIS3up+HIS3 dow2 DNA片段的线性化载体；将Km URA3和HIS3 dow1两个DNA片段利用其10-25bp同源序列，通过一步克隆法连接在pMD-18T线性化载体上；PCR扩增连接有Km URA3和HIS3 dow1片段的pMD-18T质粒，获得HIS3dow1-Km URA3 DNA片段，其中HIS3 dow1-Km URA3 DNA片段5’末端和HIS3up的3’末端有10-25bp同源序列，HIS3 dow1-Km URA3 DNA片段3’末端和HIS3 dow2的5’末端有10-25bp同源序列，HIS3 dow1-Km DNA片段和包含HIS3 up+HIS3 dow2 DNA片段的线性化载体进行一步克隆法连接，连接产物转化感受态E.coil，进行阳性克隆筛选、测序鉴定，最终构建获得包含his3Δ cassette(HIS3 up-HIS3 dow1-Km URA3-HIS3 dow2，4段DNA片段)的pMD-18T his3Δ-cas质粒。
构建pMD-18T his3Δ-cas质粒测序后比对结果，第一段是HIS3 up(SEQ IN No.18)，第二段是HIS3 dow1(SEQ IN No.19)，第三段是KmURA3(SEQ IN No.20)，第四段是HIS3 dow2(SEQ IN No.21)。
2、FIM-2(ura3Δhis3Δ)菌株筛选和验证
选用同源重组方法敲除马克思克鲁维酵母FIM-1基因组上HIS3：首先将pMD-18T his3Δ-cas质粒用BamH1和Kpn1在33.5℃进行3h酶切，酶切后回收3206bp片段，获得his3Δcassette。然后将该片段转化FIM-1(ura3Δ)菌株。
参照图9，his3Δcassette转化FIM-1(ura3Δ)菌株后，5’和3’两端的Km HIS3同源片段和基因组上的Km HIS3基因在筛选压力下发生同源重组，将突变型DNA片段置换到原基因组Km HIS3基因位置。利用Km URA3标签基因，在Uracil缺陷培养条件下筛选阳性菌落，验证鉴定后的菌株即含有Km URA3标签，可以正常表达Km URA3基因编码蛋白。再将该阳性菌株在含5-FOA药物培养条件下培养，利用药物的筛选压力，使得转化DNA片段上在Km URA3两端的Km HIS3下游565bp同源片段发生内源性同源重组，将Km URA3标签从基因组上去掉，这样就可以实现Km URA3标签的重复使用。
参照图10，将转化后菌液涂布在SC-URA平板上，30℃倒置培养48-72h后，挑单克隆分别涂布于SC-Uracil、SC-Histidine平板。30℃倒置培养12-16h后，在SC-Uracil平板生长，在SC-Histidine平板不生长的克隆即转化阳性克隆，该菌株即基因组Km HIS3位置含有Km URA3标签基因的his3Δ菌株。将该菌株涂布于YEPD+5-FOA平板，30℃倒置培养12-16h后，阳性克隆即发生了内源性同源重组，Km URA3标签基因掉落后的FIM-2(ura3Δhis3Δ)菌株，测序鉴定后的FIM-2(ura3Δhis3Δ)菌株涂布SC-Uracil、SC-Histidine平板，30℃倒置培养12-16h后都无法生长，如图10最后两幅照片所示。
敲除ura3、ade2基因马克斯克鲁维酵母工程菌株FIM-4(ura3Δade2Δ)的构建、 敲除ura3、his3、ade2基因马克斯克鲁维酵母工程菌株FIM-5(ura3ΔhisΔade2Δ) 的构建
1、KmADE2基因克隆和pMD-18T ade2Δ-cas质粒构建
克隆获得马克思克鲁维酵母FIM野生菌株ADE2基因序列。根据实验室K.marxianusFIMwt基因组测序数据，用K.latics KLLA0E02685g基因序列进行BLAST，获得3段同源序列：
＞fragment_1(上游，SEQ IN No.22，ADE2 up)
＞fragment_2(下游，SEQ IN No.23，ADE2 dow1)
＞fragment_3(下游，SEQ IN No.24，ADE2 dow2)
根据比对结果，在同源序列上游和下游分别设计引物KmAED2-F(SQE ID No.27)、KmAED2-R(SQE ID No.28)PCR扩增KmAED2，将PCR产物克隆到pMD-18T载体上，获得KmAED2序列(SEQ IN No.26)。测序结果和K.latics KLLA0E02685g基因序列进行比对，同源度为86.8％。DNA序列翻译后的蛋白氨基酸序列(SEQ IN No.25)和K.latics KLLA0E02685g蛋白氨基酸序列同源度为87％。
根据测序获得的1710bp KmADE2序列，首先在上下游分别设计引物，PCR扩增获得KmADE2序列上游722bp DNA片段(ADE2 up)、KmADE2序列下游617bp DNA片段(ADE2 dow1)和693bp DNA片段(ADE2 dow2)、以及Km URA3 1430bp标签基因序列(SEQ ID No.22：Chromosome1：1542629-1544057，1430bp)，同时将pMD-18T空载质粒用Pstl、Smal双酶切后回收获得的线性化载体。
表4，扩增KmADE2基因所用引物
ADE2 up-F 5’-cgaattcgagctcggtacccggatccgatagtggaggccgctcacagattg-3’
ADE2 up-R 5’-cctagaacatctctacaccgaaaatacc-3’
ADE2 dow1-F 5’-gagatgttctaggatttgcctatgccaaaagaattcac-3’
ADE2 dow1-R 5’-tcagaattcgtttggacgatagaatgcaaagaatc-3’
KmURA3-F 5’-atcgtccaaacgaattctgattggaaagaccattc-3’
KmURA3-R 5’-ataggcaaataggtccgagtacactcgaacctctgcttg-3’
ADE2 dow2-F 5’-cctatttgcctatgccaaaagaattcac-3’
ADE2 dow2-R 5’-cagtgccaagcttgcatgccggatccgaattctaatagctagtaaagcagc-3’
参照图11将上述各DNA片段通过优化后一步克隆法连到载体上，获得 KmURA3作为分子筛选标签的KmADE2基因敲除的中断盒pMD-18T ade2Δ-cas质粒。
首先将pMD-18T ade2Δ-cas质粒用Sph1和Kpn1，37℃进行3h酶切，酶切后回收3462bp片段。将该DNA片段转化FIM-1(ura3Δ)菌株，构建FIM-4(ura3Δade2Δ)工程菌株；将该DNA片段转化FIM-2(ura3Δhis3Δ)菌株，构建FIM-5(ura3Δhis3Δade2Δ)工程菌株。
将转化后菌液涂布在SC-Uracil平板上，30℃倒置培养48-72h后，挑单克隆分别涂布于SC-Uracil、SC-Adenine平板。30℃倒置培养12-16h后，在SC-Uracil平板生长，在SC-Adenine平板不生长的克隆即转化阳性克隆，该菌株即基因组Km ADE2位置含有Km URA3标签基因的ade2Δ菌株。将该菌株涂布于YEPD+5-FOA平板，30℃倒置培养12-16h后，阳性克隆即发生了内源性同源重组，Km URA3标签基因掉落。
挑YEPD+5-FOA平板阳性克隆于YEPD液体培养基，30℃摇床培养24-48h后，抽提基因组，PCR扩增ade2Δ片段并测序。测序后ade2Δ序列和Km ADE2序列进行比对分析，FIM-4(ura3Δade2Δ)菌株、FIM-5(ura3Δhis3Δade2Δ)菌株ade2Δ序列相比Km HIS3序列缺失120bp。
测序鉴定后的FIM-4(ura3Δade2Δ)菌株涂布SC-Uracil、SC-Adenine平板，30℃倒置培养12-16h后都无法生长(图12)。
测序鉴定后的FIM-5(ura3Δhis3Δade2Δ)菌株涂布SC-Uracil、SC-Histidine、SC-Adenine平板，30℃倒置培养12-16h后都无法生长(图13)。
敲除his3基因马克斯克鲁维酵母工程菌株FIM-3(his3Δ)的构建、建敲除his3、 ade2基因马克斯克鲁维酵母工程菌株FIM-6(his3ΔadeΔ)的构建
通过NCBI数据库检索获得K.marxianus URA3基因序列，根据获得的URA3基因序列设计引物KmURA3-F、KmURA3-R。
PCR扩增获得包含URA3 ORF共3123bp(Chromosome1：1541868-1544992，3123bp)的KmURA3 DNA片段。
将上述KmURA3 DNA片段通过LiOAc化学转化法分别转化FIM-2(ura3Δhis3Δ)菌株、FIM-5(ura3Δhis3Δade2Δ)菌株，通过同源重组回补URA3，筛选获得FIM-3(his3Δ)和FIM-6(his3Δade2Δ)工程菌株。
FIM-3(his3Δ)菌株涂布SC-Uracil平板，30℃倒置培养12-16h后生长，而涂布SC-Histidine平板，30℃倒置培养12-16h后无法生长，如图14所示。
FIM-6(his3Δade2Δ)菌株涂布SC-Uracil平板，30℃倒置培养12-16h后生长，而涂布SC-Histidine、SC-Adenine平板，30℃倒置培养12-16h后都无法生长。
实施例5，敲除菊粉酶基因
用引物F1(SEQ ID No.79)和引物R1(SEQ ID No.80)扩增克鲁维酵母基因组，该产物被命名为A片段，其中包含了菊粉酶基因上游854bp-131bp的序列。
用引物F2(SEQ ID No.81)和引物R2(SEQ ID No.82)扩增克鲁维酵母基因组，该产物被命名为B片段，其中包含了菊粉酶基因下游1bp-850bp的序列。
用引物F3(SEQ ID No.83)和引物R3(SEQ ID No.84)扩增克鲁维酵母基因组，该产物被命名为C片段，其中包含了URA3基因的启动子和ORF。
用引物F4(SEQ ID No.85)和引物R4(SEQ ID No.86)扩增克鲁维酵母基因组，该产物被命名为D片段，其中包含了菊粉酶基因331bp-1029bp的序列。
将片段A、B、C、D进行连接，连接后的片段被命名为E。将E片段转入URA3基因突变的Kluyveromyces marxianus的菌株中，在缺乏Uracil的SC培养基上筛选转化子。将阳性转化子涂布于含有5-Fluoroorotic Acid(5FOA，2g/L)的YPD平板上，获得反筛后的克隆。利用引物F5(SEQ ID No.87)和R5(SEQ ID No.88)扩增反筛后的克隆的基因组，获得的2300bp产物为敲除了菊粉酶基因的克鲁维酵母菌株。
实施例6，用于在马克斯克鲁维酵母CGMCC No.10621营养缺陷型菌株中进行 外源基因分泌表达的重组载体
重组载体PUKD112的构建
本实施例中重组载体PUKD112的构建方法如下：
步骤1，扩增pUC19质粒
利用引物(正向引物SEQ ID No.47、反向引物SEQ ID No.48)扩增pUC19 质粒。对PCR产物进行回收，该产物被命名为A片段。
步骤2，扩增Gateway系统载体的pcYGW
利用引物(正向引物SEQ ID No.49、反向引物SEQ ID No.50)扩增Gateway系统载体的pcYGW。对PCR产物进行回收，该产物被命名为B片段。
步骤3，扩增PKD1载体
利用引物(正向引物SEQ ID No.51、反向引物SEQ ID No.52)扩增PKD1载体。对PCR产物进行回收，该产物被命名为C片段。
步骤4，连接片段A、B、C
将片段A、B、C连接，转化大肠杆菌，获得的质粒命名为PUC19-PKD1。
步骤5，扩增PUC19-PKD1质粒
利用引物(正向引物SEQ ID No.53、反向引物SEQ ID No.54)扩增PUC19-PKD1质粒。对PCR产物进行回收，该产物被命名为D片段。D片段中包括了部分PUC19序列和PKD1序列。
步骤6，扩增URA3基因启动子和ORF片段
按照酵母基因组提取试剂盒(DP307)的说明书操作，利用引物(正向引物SEQ ID No.55、反向引物SEQ ID No.56)扩增马克斯克鲁维酵母的基因组。对PCR产物进行回收，该产物被命名为E片段。E片段中包含了URA3基因的启动子和ORF。
步骤7，扩增菊粉酶基因的启动子和信号肽片段
利用引物(正向引物SEQ ID No.57、反向引物SEQ ID No.58)扩增马克斯克鲁维酵母的基因组。对PCR产物进行回收，该产物被命名为F片段。F片段中包含了菊粉酶基因的启动子、信号肽和部分多克隆位点。
步骤8，扩增菊粉酶基因的终止子片段
利用引物(正向引物SEQ ID No.59、反向引物SEQ ID No.60)扩增马克斯克鲁维酵母的基因组。PCR扩增条件同步骤1，除了延伸时间为1分钟。按照Simgen公司的胶回收试剂盒的说明书操作，对PCR产物进行回收，该产物被命名为G片段。G片段中包含了菊粉酶基因的终止子和部分多克隆位点。
步骤9，连接D、E、F、G片段
将片段D、E、F、G进行连接，转化大肠杆菌，获得的质粒命名为PUKDN112。
由杰李公司对PUKDN112进行测序。参照图15，测序结果如下：
全长：11472bp
2031-1171bp：beta-lactamase(氨苄抗性基因)
3112-7870：PKD1载体序列
7879-9094：菊粉酶启动子
9095-9162：菊粉酶信号肽
9173-9192：多克隆位点，包括Smal/Xmal、Spel、Notl三个位点。
9197-10061：菊粉酶终止子
10062-10647：Km URA3启动子
10648-11451：Km URA3 ORF
PUKD112全序列如SEQ ID No.41所示。
用途：
将外源基因插入PUKDN112的多克隆位点中，利用测序确定基因序列与菊粉酶信号肽的读框一致。
转化采用LiAc法，按照参考文献进行操作(World Journal of Microbiology&Biotechnology 16：653-654，2000)。将含有外源基因的PUKDN112质粒转入URA3基因突变的马克斯克鲁维酵母CGMCC No.10621菌株中，在缺乏Uracil的培养基上筛选得到URA3+转化子。URA3+转化子可用于外源基因的分泌表达，表达和检测的方法按照参考文献进行(Appl Microbiol Biotechnol(2005)67：364-369)。
重组载体PUKD117的构建
用引物(正向引物SEQ ID No.90、反向引物SEQ ID No.91)扩增PPIC9K载体。对PCR产物进行回收，该产物被命名为A片段。A片段中包含了酿酒酵母alpha因子信号肽。
按照Agilent公司的QuikChange II试剂盒的说明书操作，A片段为引物对PUKDN112质粒进行突变，突变后的质粒为PUKDN117。由杰李公司进行测序，确定alpha信号肽的序列。
参照图16，测序结果如下：
全长：11673bp
1031-1171bp：beta-lactamase(氨苄抗性基因)
3112-7870p：PKD1载体序列
7879-9094：菊粉酶启动子
9095-9364：alpha信号肽
9374-9393：多克隆位点，包括Smal/Xmal、Spel、Notl三个位点。
9398-10262：菊粉酶终止子
10263-10848：Km URA3启动子
10849-11652：Km URA3 ORF
PUKD117全序列如SEQ ID 42所示。
重组载体PUKD115的构建
按照Agilent公司的QuikChange II试剂盒的说明书操作，对PUKDN112质粒进行突变，突变所用的引物为SEQ ID No.65和SEQ ID No.66。突变后的质粒为PUKDN115。由杰李公司进行测序，确定多克隆位点的序列。参照图17，测序结果如下：
全长：11394bp
1031-1171bp：beta-lactamase(氨苄抗性基因)
3112-7870：PKD1载体序列
7879-9094：菊粉酶启动子
9095-9114：多克隆位点，包括Spel、Smal/Xmal、Notl三个位点。
9119-9983：菊粉酶终止子
9984-10569：Km URA3启动子
10570-11373：Km URA3 ORF
PUKD115全序列如SEQ ID No.43所示。
重组载体PUKD118的构建
步骤1，用引物(正向引物SEQ ID No.67、反向引物SEQ ID No.68)扩增PUKDN112载体。对PCR产物进行回收，该产物被命名为A片段。A片段中包含了菊粉酶启动子，多克隆位点，菊粉酶终止子，URA3启动子和URA3 ORF。
步骤2，用引物(正向引物SEQ ID No.69、反向引物SEQ ID No.70)扩增PUKDN112载体。对PCR产物进行回收，该产物被命名为B片段。B片段中包 含了PKD1序列。
步骤3，获得转化子
按照NEB公司Gibson Assembly Master Mix的使用说明书操作，将片段A和B进行连接，将连接产物转化URA3基因突变的Kluyveromyces marxianus的菌株中，在缺乏uracil的培养基上筛选转化子。
根据天根的酵母基因组提取试剂盒(DP307)的说明书操作，抽提URA3+转化子的基因组。用正向引物(SEQ ID No.71)和反向引物(SEQ ID No.72)扩增基因组。PCR扩增条件与PUKD112质粒构建时步骤1条件相一致，除了延伸时间为1分钟。按照Simgen的胶回收试剂盒说明书操作，对PCR产物进行回收，该产物被命名为C片段。该片段中包含了A片段和B片段的连接部分。由杰李公司对片段C进行测序，验证A片段和B片段是否正确连接。参照图18，测序结果如下：
全长：8357bp
1-4759：PKD1载体序列
4768-5983：菊粉酶启动子
5984-6051：alpha信号肽
6062-6081：多克隆位点，包括Smal/Xmal、Spel、Notl三个位点。
6086-6950：菊粉酶终止子
6951-7536：Km URA3启动子
7537-8340：Km URA3 ORF
PUKD118全序列如SEQ ID No.44所示。
按照Zymoresearch公司的Zymoprep Yeast Plasmid Miniprep II试剂盒的说明书操作，对含有正确连接产物的转化子进行酵母质粒的抽提，获得质粒为PUKDN118。
用途：
用正向引物SEQ ID No.73和反向引物SEQ ID No.74扩增PUKD118质粒。获得的PCR产物被命名为D片段。
在扩增外源基因ORF的正向引物的5’端添加SEQ ID No.75的序列，在扩增外源基因ORF的反向引物的5’端添加SEQ ID No.76的序列。利用该对引物 扩增外源基因。获得的PCR产物被命名为E片段。
按照NEB公司Gibson Assembly Master Mix的使用说明书操作，将片段D和E进行连接，将连接产物转化URA3基因突变的Kluyveromyces marxianus的菌株中，在缺乏uracil的培养基上筛选转化子。
按照Zymoresearch公司的Zymoprep Yeast Plasmid Miniprep II试剂盒的说明书，对URA3+转化子进行酵母质粒的抽提。用正向引物Fp(SEQ ID No.77)和反向引物Rp(SEQ ID No.78)扩增质粒。按照Simgen的胶回收试剂盒对PCR产物进行回收，该产物被命名为F片段。由杰李公司对片段F进行测序，验证外源基因与PUKD118是否连接正确。
选择含有正确连接了外源基因的PUKD118的转化子，进行外源基因的分泌表达，表达和检测的方法按照参考文献进行(Appl Microbiol Biotechnol(2005)67：364-369)。
重组载体PUKD114的构建
突变PUKDN112，按照Agilent公司的QuikChange II试剂盒的说明书操作，对PUKDN112质粒进行突变。突变所用引物为SEQ ID No.61和SEQ ID No.62。突变后的质粒即为PUKDN114。由杰李公司进行测序，确定六聚组氨酸标签(6His Tag)序列和多克隆位点。参照图19，测序结果如下：
全长：11415bp
2031-1171bp：beta-lactamase(氨苄抗性基因)
3112-7870：PKD1载体序列
7879-9094：菊粉酶启动子
9098-9115：6 His Tag
9116-9135：多克隆位点，包括Spel、Smal/Xmal、Notl三个位点。
9140-10004：菊粉酶终止子
10005-10590：Km URA3启动子
10591-11394：Km URA3 ORF
PUKD112全序列如SEQ ID No.45所示。
重组载体PUKD116的构建
突变PUKDN112，按照Agilent公司的QuikChange II试剂盒的说明书操作， 对PUKDN112质粒进行突变。突变所用引物为SEQ ID No.63和SEQ ID No.64。突变后的质粒即为PUKDN116。由杰李公司进行测序，确定6 His Tag序列和多克隆位点。参照图20，测序结果如下：
全长：11412bp
1031-1171bp：beta-lactamase(氨苄抗性基因)
3112-7870p：PKD1载体序列
7879-9094：菊粉酶启动子
9095-9114：多克隆位点，包括Spel、Smal/Xmal、Notl三个位点。
9116-9133：6 His Tag
9137-10001：菊粉酶终止子
10002-10587：KmURA3启动子
10588-11391：KmURA3 ORF
PUKD116全序列如SEQ ID 46所示。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (16)

  1. 一种马克斯克鲁维酵母菌(Kluyveromyces marxianus)，其特征在于，该马克斯克鲁维酵母菌具有SEQ ID No.1所述基因序列、或与SEQ ID No.1同源度至少为90％的基因序列。
  2. 根据权利要求1所述的马克斯克鲁维酵母菌，其特征在于，该马克思克鲁维酵母菌保藏号为CGMCC No.10621。
  3. 一种权利要求1所述马克思克鲁维酵母构建的重组蛋白表达系统。
  4. 根据权利要求3所述的重组蛋白表达系统，其特征在于，是权利要求1所述马克思克鲁维酵母菌株敲除全部或部分营养基因之后的营养缺陷型菌株，或者权利要求1所述马克思克鲁维酵母菌株敲除菊粉酶基因的菌株，或者所述营养缺陷型菌株敲除菊粉酶基因的菌株。
  5. 根据权利要求3或4所述的马克思克鲁维酵母构建的重组蛋白表达系统，其特征在于，所述营养基因选自URA3基因、HIS3基因、ADE2基因中的任意一种或几种。
  6. 一种用于在权利要求1所述马克思克鲁维酵母构建的重组蛋白表达系统、或权利要求3所述重组蛋白表达系统中进行外源基因分泌表达的重组载体，其特征在于，所述重组载体选自如下重组载体A)-C)中的任意一种或几种：
    重组载体A)：按照顺序包括PKD1载体、菊粉酶启动子、信号肽、多克隆位点、菊粉酶终止子、营养基因启动子、营养基因开放阅读框(ORF)；
    重组载体B)：按照顺序包括PKD1载体、菊粉酶启动子、多克隆位点、菊粉酶终止子、营养基因启动子、营养基因开放阅读框(ORF)；
    重组载体C)：按照顺序包括PKD1载体序列、菊粉酶启动子、多克隆位点、菊粉酶终止子、营养基因启动子、营养基因开放阅读框(ORF)；其中，所述重组载体还包括多聚组氨酸标签，所述多聚组氨酸标签位于多克隆位点的C端或N端。
  7. 根据权利要求6所述的重组载体，其特征在于，所述重组载体不包括氨苄抗性基因。
  8. 根据权利要求6所述的重组载体，其特征在于，所述信号肽选自菊粉酶信号 肽、或酵母alpha因子信号肽中的任意一种或几种。
  9. 一种转化子，其特征在于，所述转化子选自：
    A)权利要求6所述任意一种或几种重组载体转入权利要求1所述马克斯克鲁维酵母的菌株中；
    B)权利要求6所述任意一种或几种重组载体转入权利要求3所述重组蛋白表达系统中。
  10. 一种权利要求1所述马克斯克鲁维酵母、或权利要求3所述重组蛋白表达系统、或权利要求9所述的转化子的应用，其特征在于，所述马克斯克鲁维酵母、或所述重组蛋白表达系统、或所述转化子用作动物饲料添加剂和/或动物饲料、食品、疫苗或药物的发酵菌剂。
  11. 根据权利要求10所述的马克斯克鲁维酵母的应用，其特征在于，
    所述发酵菌剂直接添加至动物饲料添加剂和/或动物饲料、食品、疫苗或药物中，或者
    所述发酵菌剂对原料进行发酵制备含有发酵产物的动物饲料添加剂和/或动物饲料、食品、疫苗或药物，或者
    所述发酵菌剂表达的含有酶或蛋白的发酵物添加至动物饲料添加剂和/或动物饲料、食品、疫苗或药物中。
  12. 根据权利要求10或12所述的应用，其特征在于，所述应用选自：使饲料具有提高动物免疫力、和/或提高动物消化能力、和/或保护动物不受自由基损伤、和/或生产更多营养成分的能力。
  13. 一种动物饲料、食品、疫苗或药物添加剂，其特征在于，所述动物饲料添加剂至少包括权利要求1所述马克斯克鲁维酵母及其表达酶/蛋白、或权利要求3所述重组蛋白表达系统及其表达酶/蛋白、或权利要求9所述的转化子及其表达酶/蛋白中的任意一种或几种。
  14. 一种产品，所述产品选自动物饲料、食品、疫苗或药物中的任意一种或几种，其特征在于，包括至少一种添加剂，所述至少一种添加剂中包括权利要求1所述马克斯克鲁维酵母及其表达酶/蛋白、或权利要求3所述重组蛋白表达系统及其表达酶/蛋白、或权利要求9所述的转化子及其表达酶/蛋白中的任意一种或几种。
  15. 一种发酵的产品，所述产品选自动物饲料、食品、疫苗或药物中的任意一种 或几种，其特征在于，所述发酵的产物是原料在发酵菌剂存在下发酵得到，其中，所述发酵菌剂至少包括权利要求1所述马克斯克鲁维酵母、或权利要求3所述重组蛋白表达系统、或权利要求9所述的转化子。
  16. 根据权利要求13所述的添加剂、或权利要求14-15任意一项所述产品，其特征在于，还包括营养元素、调味剂、其他有益菌、防霉剂、胆汁酸、螯合肽、复合酶、促生长组分、抗生素、色素、以及其他可用于所述添加剂的组分中的任意一种或几种。
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